Transcriptomics of Besnoitia besnoiti-Infected Fibroblasts Reveals Hallmarks of Early Fibrosis and Cancer Progression.

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, TGFß1, FOSB). We have identified potential biomarkers and host pathways regulated during fibrosis that may hold prognostic significance and could emerge as potential therapeutic targets.

Preliminary investigations of parasite contamination of water sources in Armenia.

The intestinal protozoan parasites, Cryptosporidium and Giardia, are known to have a global distribution, infecting and causing disease in a range of hosts, including people, livestock, pets, and wildlife. However, data from some regions is very sparse. In Armenia, in the Caucasus region of West Asia, only scanty data are available, with just a few surveys on Cryptosporidium infections in livestock, and no available data on human infections or environmental contamination. As part of implementation of water analysis methods for these parasites in Armenia, 24 raw water samples and two sediment samples were analysed for these parasites using a range of approaches, including modified Ziehl-Neelsen, Lugol stain, immunofluorescent antibody test (IFAT), qPCR and, on sediment samples, immunomagnetic separation and IFAT. Results suggest substantial contamination of raw water sources and indicate the need for further targeted studies using appropriate methods and collecting data on host infections in catchment areas.

Gene expression profile of HCT-8 cells following single or co-infections with Cryptosporidium parvum and bovine coronavirus.

Among the causative agents of neonatal diarrhoea in calves, two of the most prevalent are bovine coronavirus (BCoV) and the intracellular parasite Cryptosporidium parvum. Although several studies indicate that co-infections are associated with greater symptom severity, the host-pathogen interplay remains unresolved. Here, our main objective was to investigate the modulation of the transcriptome of HCT-8 cells during single and co-infections with BCoV and C. parvum. For this, HCT-8 cells were inoculated with (1) BCoV alone, (2) C. parvum alone, (3) BCoV and C. parvum simultaneously. After 24 and 72 h, cells were harvested and analyzed using high-throughput RNA sequencing. Following differential expression analysis, over 6000 differentially expressed genes (DEGs) were identified in virus-infected and co-exposed cells at 72 hpi, whereas only 52 DEGs were found in C. parvum-infected cells at the same time point. Pathway (KEGG) and gene ontology (GO) analysis showed that DEGs in the virus-infected and co-exposed cells were mostly associated with immune pathways (such as NF-κB, TNF-α or, IL-17), apoptosis and regulation of transcription, with a more limited effect exerted by C. parvum. Although the modulation observed in the co-infection was apparently dominated by the virus, over 800 DEGs were uniquely expressed in co-exposed cells at 72 hpi. Our findings provide insights on possible biomarkers associated with co-infection, which could be further explored using in vivo models.

Phase Angle in Head and Neck Cancer: A Sex-Differential Analysis from Biological and Clinical Behavior to Health-Related Quality of Life.

Head and neck cancer (H&NC) is a diverse category of tumors related to malignancies in the common aerodigestive pathway, with high metabolic rate, poor nutritional and treatment outcomes, and elevated mortality despite the best standard treatment. Herein, we focus on determining how the phase angle (PA) differs across sex as a predictor of poor prognosis, low quality-of-life (QoL) scores, and mortality in patients with head and neck cancer. This follow-up study presents a sex-differential analysis in a prospective cohort of 139 head and neck cancer patients categorized by sex as male (n = 107) and female (n = 32). Patients were compared in terms of nutritional, biochemical, and quality-of-life indicators between low and normal PA in women (<3.9° (n = 14, 43.75%) and ≥3.9°) and men (<4.5° (n = 62, 57.9%) and ≥4.5°). Our results show that most patients were in locally advanced clinical stages (women: n = 21 (65.7%); men: n = 67 (62.6%)) and that patients with low PA had a lower punctuation in parameters such as handgrip strength, four-meter walking speed, albumin, C-reactive protein (CRP), and CRP/albumin ratio (CAR), as well as the worst QoL scores in functional and symptomatic scales in both the male and female groups. A comparison between sexes revealed significant disparities; malnourishment and tumor cachexia related to an inflammatory state was more evident in the women's group.

Transcriptional changes associated with apoptosis and type I IFN underlie the early interaction between Besnoitia besnoiti tachyzoites and monocyte-derived macrophages.

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.

Nothrus mite as a potential cause of exacerbation of pruritus in a cat.

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.

Bovine Enteroids as an In Vitro Model for Infection with Bovine Coronavirus.

Bovine coronavirus (BCoV) is one of the major viral pathogens of cattle, responsible for economic losses and causing a substantial impact on animal welfare. Several in vitro 2D models have been used to investigate BCoV infection and its pathogenesis. However, 3D enteroids are likely to be a better model with which to investigate host-pathogen interactions. This study established bovine enteroids as an in vitro replication system for BCoV, and we compared the expression of selected genes during the BCoV infection of the enteroids with the expression previously described in HCT-8 cells. The enteroids were successfully established from bovine ileum and permissive to BCoV, as shown by a seven-fold increase in viral RNA after 72 h. Immunostaining of differentiation markers showed a mixed population of differentiated cells. Gene expression ratios at 72 h showed that pro-inflammatory responses such as IL-8 and IL-1A remained unchanged in response to BCoV infection. Expression of other immune genes, including CXCL-3, MMP13, and TNF-α, was significantly downregulated. This study shows that the bovine enteroids had a differentiated cell population and were permissive to BCoV. Further studies are necessary for a comparative analysis to determine whether enteroids are suitable in vitro models to study host responses during BCoV infection.

Phase Angle, a Cornerstone of Outcome in Head and Neck Cancer.

In patients with head and neck cancer, malnutrition is common. Most cases are treated by chemo-radiotherapy and surgery, with adverse effects on the aerodigestive area. Clinical and biochemical characteristics, health-related quality of life, survival, and risk of death were studied. The selected subjects were divided into normal- and low-phase-angle (PA) groups and followed up for at least two years. Mean ages were 67.2 and 59.3 years for low and normal PA, respectively. Patients with PA < 4.42° had significant differences in age, anthropometric and biochemical indicators of malnutrition, and inflammatory status compared to patients with PA > 4.42°. Statistical differences were found in the functional and symptom scales, with lower functional scores and higher symptom scores in patients with low PA. Median survival was 19.8 months for those with PA < 4.42° versus 34.4 months for those with PA > 4.42° (p < 0.001).The relative risk of death was related to low PA (2.6; p < 0.001). The percentage of living patients (41.7%) is almost the same as the percentage of deceased subjects (43.1%; p = 0.002), with high death rates in patients with PA < 4.42°. Phase angle was the most crucial predictor of survival and a risk factor for death in the studied cases.

Etiology and Risk Factors for Admission to the Pediatric Intensive Care Unit in Children With Encephalitis in a Developing Country.

OBJECTIVE: To describe a cohort of pediatric patients with encephalitis and their risk factors for admission to the pediatric intensive care unit (PICU). STUDY DESIGN: Children (<18 years old), with encephalitis evaluated by conventional microbiology and syndromic, multiplex test in cerebrospinal fluid (CSF) between July 2017 and July 2020, were recruited from 14 hospitals that comprise the Colombian Network of Encephalitis in Pediatrics. Multivariate analyses were used to evaluate risk factors associated with the need for PICU admission. RESULTS: Two hundred two children were included, of which 134 (66.3%) were male. The median age was 23 months (IQR 5.7-73.2). The main etiologies were bacteria (n = 55, 27%), unspecified viral encephalitis (n = 44, 22%) and enteroviruses (n = 27, 13%), with variations according to age group. Seventy-eight patients (38.6%) required management in the PICU. In multivariate analysis, factors associated with admission to the PICU were the presence of generalized seizures (OR 2.73; 95% CI: 1.82-4.11), status epilepticus (OR 3.28; 95% CI: 2.32-4.62) and low leukocyte counts in the CSF (OR 2.86; 95% CI: 1.47-5.57). Compared with enterovirus, bacterial etiology (OR 7.50; 95% CI: 1.0-56.72), herpes simplex encephalitis (OR 11.81; 95% CI: 1.44-96.64), autoimmune encephalitis (OR 22.55; 95% CI: 3.68-138.16) and other viral infections (OR 5.83; 95% CI: 1.09-31.20) increased the risk of PICU admission. CONCLUSIONS: Data from this national collaborative network of pediatric patients with encephalitis allow early identification of children at risk of needing advanced care and can guide the risk stratification of admission to the PICU.

Quality of Life and Functionality of Head and Neck Cancer Patients Are Diminished As a Function of Sarcopenia and Obesity.

BACKGROUND: Health-related quality of life (QoL) is a measure that allows us to know the patient's perception of well-being and how it is affected by their disease and treatments. In cancer patients, sarcopenia has been associated with low scores on various instruments used to assess the QoL; however, little information is available on the effects of sarcopenia and sarcopenic obesity on the QoL of patients with head and neck cancer (H&NC). METHODS: In this cross-sectional study with 71 H&NC patients aged between 40 and 80 years, we describe the scores on the instruments EORTC QLQ C-30 and EORTC QLQ-H&N35 according to the sarcopenia phenotype (NSG, nonsarcopenic group; SG, sarcopenic group; and SOG, sarcopenic obesity group), hand-grip strength, gait speed, total lymphocyte count, albumin, cholesterol and C-reactive protein, and the relationships between these variables. RESULTS: The prevalence of sarcopenia and sarcopenic obesity was 48% and 28%, respectively. The QoL analysis showed that NSG had higher scores on the physical functioning scale [NSG 93 (83-100); SG 73 (52-88); SOG 83 (53-93), P = .009] and lower scores on the fatigue scale [NSG 11 (0-22); S 39 (30-67); SOG 44 (14-56); P = .004]. The NSG had a higher hand-grip strength (31.1 kg) than SG (24.1 kg, P = .007) and SOG (26.3 kg, P = .001), and a lower C-reactive protein. The SG and SOG showed no differences between them. CONCLUSIONS: Patients with sarcopenia or sarcopenic obesity have lower physical performance and a higher level of fatigue than nonsarcopenic patients. This loss of function can maintain or worsen sarcopenia due to the patient's self-restraint in physical exertion that encourages an increase in muscle tissue.

Interactions between Cryptosporidium parvum and bovine corona virus during sequential and simultaneous infection of HCT-8 cells.

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.

Maternal and Foetal Cellular Immune Responses in Dams Infected With High- and Low- Virulence Isolates of Neospora caninum at Mid-Gestation.

Bovine neosporosis is currently considered one of the main causes of abortion in cattle worldwide and the outcome of the infection is, in part, determined by Neospora caninum isolate virulence. However, the dam and foetal immune responses associated with this factor are largely unknown. We used a model of bovine infection at day 110 of gestation to study the early infection dynamics (10- and 20-days post-infection, dpi) after experimental challenge with high- and low-virulence isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively). In the present work, dam peripheral cellular immune responses were monitored twice a week from -1 to 20 dpi. At different time points, IFN-γ and IL-4 production was investigated in stimulated dam blood and the percentage of monocytes, NK cells, B cells and T cells (CD4+, CD8+ and γδ) in peripheral blood mononuclear cells (PBMC) were determined by flow cytometry. In addition, maternal iliofemoral lymph nodes and foetal spleen and thymus were collected at 10 and 20 dpi for the study of the same cell subpopulations. Peripheral immune response dynamics were similar after the infection with both isolates, with a significant increase in the percentage of CD4+ T cells at 6 and 9 dpi in PBMC, coincident with the higher levels of IFN-γ and IL-4 release. However, the levels of IFN-γ were significantly higher and an increase in CD8+ T cells at 9, 13 and 20 dpi was observed in the dams infected with Nc-Spain7. Nc-Spain1H infection induced higher IL4 levels in stimulated blood and a higher CD4+/CD8+ ratio in PBMC. The analysis of the maternal iliofemoral lymph node showed a significant enhancement in the percentage of NK, CD4+ and CD8+ T cells for the animals infected with the highly virulent isolate and euthanized at 20 dpi. Regarding the foetal responses, the most remarkable result was an increase in the percentage of monocytes at 20 dpi in the spleen of foetuses from both infected groups, which suggests that foetuses were able to respond to N. caninum infection at mid gestation. This work provides insights into how isolate virulence affects the maternal and foetal immune responses generated against N. caninum, which may influence the course of infection.

Identification of molecular biomarkers associated with disease progression in the testis of bulls infected with Besnoitia besnoiti.

Breeding bulls infected with Besnoitia besnoiti may develop sterility during either acute or chronic infection. The aim of this study was to investigate the molecular pathogenesis of B. besnoiti infection with prognosis value in bull sterility. Accordingly, five well-characterized groups of naturally and experimentally infected males were selected for the study based on clinical signs and lesions compatible with B. besnoiti infection, serological results and parasite detection. A broad panel of molecular markers representative of endothelial activation and fibrosis was investigated and complemented with a histopathological approach that included conventional histology and immunohistochemistry. The results indicated the predominance of an intense inflammatory infiltrate composed mainly of resident and recruited circulating macrophages and to a lesser extent of CD3+ cells in infected bulls. In addition, a few biomarkers were associated with acute, chronic or subclinical bovine besnoitiosis. The testicular parenchyma showed a higher number of differentially expressed genes in natural infections (acute and chronic infections) versus scrotal skin in experimental infections (subclinical infection). In subclinical infections, most genes were downregulated except for the CCL24 and CXCL2 genes, which were upregulated. In contrast, the acute phase was mainly characterized by the upregulation of IL-1α, IL-6 and TIMP1, whereas in the chronic phase, the upregulation of ICAM and the downregulation of MMP13, PLAT and IL-1α were the most relevant findings. Macrophages could be responsible for the highest level of gene regulation in the testicular parenchyma of severely affected and sterile bulls, and all these genes could be prognostic markers of sterility.

Micronucleated erythrocytes in peripheral blood from neonate rats fed by nursing mothers exposed to X-rays.

Most women with breast cancer can become pregnant and give birth while undergoing radiation therapy and breastfeeding is generally not contraindicated. The induction of long-lived reactive species in proteins, such as casein by X-ray radiation and DNA damage to unexposed organisms, has been shown when ingesting irradiated cheese. To determine whether exposing lactating rats to X-rays increases the number of micronucleated erythrocytes (MNEs) in peripheral blood of their unexposed or breastfeeding rat pups, 15 female Wistar rats were divided into three groups: Negative control; Experimental group exposed to X-rays, and group exposed to X-rays plus vitamin C. The mothers of groups 2 and 3 were irradiated for three consecutive days after giving birth, returning them to their respective cages each time to continue lactation. A blood sample was taken from the mothers and pups at 0, 24, and 48 hr. Blood smears were stained with acridine orange to analyze MNEs. In mother rats, the frequency of micronucleated polychromatic erythrocytes (MNPCEs) increased significantly at 24 and 48 hr in both study groups exposed to radiation. Likewise, in rat pups the MNPCE and MNE frequencies increased in both groups with radiation and radiation plus vitamin C at 24 and 48 hr, and a protection from vitamin C was observed. In conclusion, the genotoxic damage produced in rat pups that were lactated by mothers irradiated with X-rays is possibly due to the effect of long-lived reactive species that were formed in the breast milk of female Wistar rats during the irradiation process.

RNA-Seq Analyses Reveal That Endothelial Activation and Fibrosis Are Induced Early and Progressively by Besnoitia besnoiti Host Cell Invasion and Proliferation.

The pathogenesis of bovine besnoitiosis and the molecular bases that govern disease progression remain to be elucidated. Thus, we have employed an in vitro model of infection based on primary bovine aortic endothelial cells (BAEC), target cells during the acute infection. Host-parasite interactions were investigated by RNA-Seq at two post-infection (pi) time points: 12 hpi, when tachyzoites have already invaded host cells, and 32 hpi, when tachyzoites have replicated for at least two generations. Additionally, the gene expression profile of B. besnoiti tachyzoites was studied at both pi time points. Up to 446 differentially expressed B. taurus genes (DEGs) were found in BAEC between both pi time points: 249 DEGs were up-regulated and 197 DEGs were down-regulated at 32 hpi. Upregulation of different genes encoding cytokines, chemokines, leukocyte adhesion molecules predominantly at 12 hpi implies an activation of endothelial cells, whilst upregulation of genes involved in angiogenesis and extracellular matrix organization was detected at both time points. NF-κB and TNF-α signaling pathways appeared to be mainly modulated upon infection, coordinating the expression of several effector proteins with proinflammatory and pro-fibrotic phenotypes. These mediators are thought to be responsible for macrophage recruitment setting the basis for chronic inflammation and fibrosis characteristic of chronic besnoitiosis. Angiogenesis regulation also predominated, and this multistep process was evidenced by the upregulation of markers involved in both early (e.g., growth factors and matrix metalloproteinases) and late steps (e.g., integrins and vasohibin). Besnoitia besnoiti ortholog genes present in other Toxoplasmatinae members and involved in the lytic cycle have shown to be differentially expressed among the two time points studied, with a higher expression at 32 hpi (e.g., ROP40, ROP5B, MIC1, MIC10). This study gives molecular clues on B. besnoiti- BAECs interaction and shows the progression of type II endothelial cell activation upon parasite invasion and proliferation.

Vascular wall injury and inflammation are key pathogenic mechanisms responsible for early testicular degeneration during acute besnoitiosis in bulls.

BACKGROUND: Bovine besnoitiosis, caused by the apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that notably impairs fertility. Acutely infected bulls may develop respiratory signs and orchitis, and sterility has been reported in chronic infections. However, the pathogenesis of acute disease and its impact on reproductive function remain unknown. METHODS: Herein, we studied the microscopic lesions as well as parasite presence and load in the testis (pampiniform plexus, testicular parenchyma and scrotal skin) of seven bulls with an acute B. besnoiti infection. Acute infection was confirmed by serological techniques (IgM seropositive results and IgG seronegative results) and subsequent parasite detection by PCR and histological techniques. RESULTS: The most parasitized tissue was the scrotal skin. Moreover, the presence of tachyzoites, as shown by immunohistochemistry, was associated with vasculitis, and three bulls had already developed juvenile tissue cysts. In all animals, severe endothelial injury was evidenced by marked congestion, thrombosis, necrotizing vasculitis and angiogenesis, among others, in the pampiniform plexus, testicular parenchyma and scrotal skin. Vascular lesions coexisted with lesions characteristic of a chronic infection in the majority of bulls: hyperkeratosis, acanthosis and a marked diffuse fibroplasia in the dermis of the scrotum. An intense inflammatory infiltrate was also observed in the testicular parenchyma accompanied by different degrees of germline atrophy in the seminiferous tubules with the disappearance of various strata of germ cells in four bulls. CONCLUSIONS: This study confirmed that severe acute besnoitiosis leads to early sterility that might be permanent, which is supported by the severe lesions observed. Consequently, we hypothesized that testicular degeneration might be a consequence of (i) thermoregulation failure induced by vascular lesions in pampiniform plexus and scrotal skin lesions; (ii) severe vascular wall injury induced by the inflammatory response in the testis; and (iii) blood-testis barrier damage and alteration of spermatogenesis by immunoresponse.

A model for chronic bovine besnoitiosis: Parasite stage and inoculation route are key factors.

In this work, an experimental model for chronic besnoitiosis in bovine was developed and characterized. Using a previously established calf model, two new variables (parasite stage and inoculation route) were combined and used. Twelve Holstein Friesian 3-month-old male calves were randomly divided into four groups of three animals each. Bradyzoites were obtained from a chronically infected bull and used for inoculation via three different inoculation routes. Three groups were inoculated with 106 bradyzoites by intravenous (G1), subcutaneous (G2) and intradermal (G3) routes, and a non-infected control group (G4) was inoculated with PBS. The trial lasted for 90 days and included daily clinical monitoring as well as weekly skin biopsies and blood sampling. Sera were obtained to analyse both cellular and humoral responses. Once the calves were euthanized, tissues from the skin, eyes, respiratory and reproductive tracts, among others, were collected to study presence of the parasite. Clinically, the infection was classified as mild to moderate for the acute stage since all infected calves showed lymphadenopathy from four days post-infection (pi) and fever from one week pi until 24 days pi. However, the most relevant results were achieved during the chronic stage that was classified as moderate to severe. In fact, pathognomonic conjunctival cysts were observed in all infected calves from 40 days pi onwards and were more abundant in G3. Moreover, one calf from this group developed skin lesions (49 days pi). The microscopic tissue cysts and Besnoitia DNA were detected primarily in skin, reproductive tract and respiratory tissue samples, and parasite load was higher in G3. In conclusion, the parasite stage (bradyzoite) and the inoculation route are key factors that influence the outcome of an infection. In particular, the intradermal route led to more severe clinical signs of the chronic phase in the inoculated calves.

Lytic cycle of Besnoitia besnoiti tachyzoites displays similar features in primary bovine endothelial cells and fibroblasts.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Repurposing of commercially available anti-coccidials identifies diclazuril and decoquinate as potential therapeutic candidates against Besnoitia besnoiti infection.

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.

Effect of parasite dose and host age on the infection with Besnoitia besnoiti tachyzoites in cattle.

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.