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.
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.
Abstract
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.
In 2021, the Norwegian Scientific Committee for Food and Environment published a multi-criteria risk ranking of 20 potentially food-borne pathogens in Norway. The pathogens ranked included five ...parasite taxa (3 species, one genus, one family): Toxoplasma gondii, Echinococcus multilocularis, Giardia duodenalis, Cryptosporidium spp., and Anisakidae. Two of these, T. gondii and E. multilocularis, scored very highly (1st and 3rd place, respectively), Cryptosporidium was about midway (9th place), and G. duodenalis and Anisakidae ranked relatively low (15th and 20th place, respectively). Parasites were found, on average, more likely to present an increasing food-borne disease burden in the future than the other pathogens. Here, we review the current impact of these five potentially food-borne parasites in Norway, and factors of potential importance in increasing their future food-borne disease burden. Climate change may affect the contamination of water and fresh produce with transmission stages of the first four parasites, potentially leading to increased infection risk. Alterations in host distribution (potentially due to climate change, but also other factors) may affect the occurrence and distribution of Toxoplasma, Echinococcus, and Anisakidae, and these, coupled with changes in food consumption patterns, could also affect infection likelihood. Transmission of food-borne pathogens is complex, and the relative importance of different pathogens is affected by many factors and will not remain static. Further investigation in, for example, ten-years’ time, could provide a different picture of the relative importance of different pathogens. Nevertheless, there is clearly the potential for parasites to exert a greater risk to public health in Norway than currently occurs.
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•Risk-ranking of food-borne pathogens in Norway included 5 parasites.•Two parasites, Toxoplasma gondii and Echinococcus multilocularis, scored highly.•Increasing disease burden was, on average, higher for parasites than other pathogens.•Relevant factors include climate change, host distribution, and food consumption trends.
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.
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.
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.
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.
•Information on Cryptosporidium and Giardia in Armenia is sparse.•We investigated raw water and sediment samples collected from across Armenia.•Detection techniques included conventional staining, IFAT, and qPCR.•High detection suggests the need for investigation of both parasites in Armenia.
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.
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.
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.
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.
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.
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