Days Post-infection Research Articles

The role of Aeromonas genotyping in virulence for Dicentrarchus labrax.

Aeromonas septicemia still represents a serious challenge facing the global aquaculture sector. In the present study, Aeromonas caviae and A. veronii were isolated from four diseased European seabass (Dicentrarchus labrax) farms experiencing a high mortality rate. Diseased fish showed haemorrhages on the external body surface with exophthalmia, cataracts, scale desquamation, skin ulcers and fin erosions. The most common post-mortem findings were congested internal organs, particularly the liver and posterior kidney. Twenty-eight A. Veronii and 11 A. caviae isolates were identified biochemically by the Vitek 2 system and then confirmed by PCR and phylogenetic analysis. Hemolysin (hlyA) and aerolysin (aer) were the most abundant virulence genes in the recovered isolates, followed by cytotoxic enterotoxin (act) and heat-stable enterotoxin (ast). A. caviae was more virulent than A. veronii for D. labrax fingerlings as LD50 ranging between (>1 × 108 -6.2 × 107 ) for A. veronii and (2.9 × 107 -8.3 × 107 ) for A. caviae. The sensitivity test indicated the effectiveness of norfloxacin, doxycycline and oxytetracycline against the tested isolates. Serum cortisol significantly increased in the infected groups, while catalase and glutathione peroxidase activities significantly decreased at 2 days post-infection (DPI) and then increased at 6 DPI. The presence of virulence genes was associated with bacterial pathogenicity expressed in fish mortality rate. Virulence genes also drastically affect cortisol levels more than catalase and glutathione peroxidase levels.

Control of maternal Zika virus infection during pregnancy is associated with lower antibody titers in a macaque model.

Zika virus (ZIKV) infection during pregnancy results in a spectrum of birth defects and neurodevelopmental deficits in prenatally exposed infants, with no clear understanding of why some pregnancies are more severely affected. Differential control of maternal ZIKV infection may explain the spectrum of adverse outcomes. Here, we investigated whether the magnitude and breadth of the maternal ZIKV-specific antibody response is associated with better virologic control using a rhesus macaque model of prenatal ZIKV infection. We inoculated 18 dams with an Asian-lineage ZIKV isolate (PRVABC59) at 30-45 gestational days. Plasma vRNA and infectious virus kinetics were determined over the course of pregnancy, as well as vRNA burden in the maternal-fetal interface (MFI) at delivery. Binding and neutralizing antibody assays were performed to determine the magnitude of the ZIKV-specific IgM and IgG antibody responses throughout pregnancy, along with peptide microarray assays to define the breadth of linear ZIKV epitopes recognized. Dams with better virologic control (n= 9) cleared detectable infectious virus and vRNA from the plasma by 7 days post-infection (DPI) and had a lower vRNA burden in the MFI at delivery. In comparison, dams with worse virologic control (n= 9) still cleared detectable infectious virus from the plasma by 7 DPI but had vRNA that persisted longer, and had higher vRNA burden in the MFI at delivery. The magnitudes of the ZIKV-specific antibody responses were significantly lower in the dams with better virologic control, suggesting that higher antibody titers are not associated with better control of ZIKV infection. Additionally, the breadth of the ZIKV linear epitopes recognized did not differ between the dams with better and worse control of ZIKV infection. Thus, the magnitude and breadth of the maternal antibody responses do not seem to impact maternal virologic control. This may be because control of maternal infection is determined in the first 7 DPI, when detectable infectious virus is present and before robust antibody responses are generated. However, the presence of higher ZIKV-specific antibody titers in dams with worse virologic control suggests that these could be used as a biomarker of poor maternal control of infection and should be explored further.

Salmonella enterica induces biogeography-specific changes in the gut microbiome of pigs.

Swine are a major reservoir of an array of zoonotic Salmonella enterica subsp. enterica lineage I serovars including Derby, Typhimurium, and 4,[5],12:i:- (a.k.a. Monophasic Typhimurium). In this study, we assessed the gastrointestinal (GI) microbiome composition of pigs in different intestinal compartments and the feces following infection with specific zoonotic serovars of S. enterica (S. Derby, S. Monophasic, and S. Typhimurium). 16S rRNA based microbiome analysis was performed to assess for GI microbiome changes in terms of diversity (alpha and beta), community structure and volatility, and specific taxa alterations across GI biogeography (small and large intestine, feces) and days post-infection (DPI) 2, 4, and 28; these results were compared to disease phenotypes measured as histopathological changes. As previously reported, only S. Monophasic and S. Typhimurium induced morphological alterations that marked an inflammatory milieu restricted to the large intestine in this experimental model. S. Typhimurium alone induced significant changes at the alpha- (Simpson's and Shannon's indexes) and beta-diversity levels, specifically at the peak of inflammation in the large intestine and feces. Increased community dispersion and volatility in colonic apex and fecal microbiomes were also noted for S. Typhimurium. All three Salmonella serovars altered community structure as measured by co-occurrence networks; this was most prominent at DPI 2 and 4 in colonic apex samples. At the genus taxonomic level, a diverse array of putative short-chain fatty acid (SCFA) producing bacteria were altered and often decreased during the peak of inflammation at DPI 2 and 4 within colonic apex and fecal samples. Among all putative SCFA producing bacteria, Prevotella showed a broad pattern of negative correlation with disease scores at the peak of inflammation. In addition, Prevotella 9 was found to be significantly reduced in all Salmonella infected groups compared to the control at DPI 4 in the colonic apex. In conclusion, this work further elucidates that distinct swine-related zoonotic serovars of S. enterica can induce both shared (high resilience) and unique (altered resistance) alterations in gut microbiome biogeography, which helps inform future investigations of dietary modifications aimed at increasing colonization resistance against Salmonella through GI microbiome alterations.

Animal efficacy study of a plant extract complex (BEN815) as a potential treatment for COVID-19.

In a short time, several types of injectable and oral therapeutics have been developed and used to effectively manage patients with coronavirus disease 2019 (COVID-19). BEN815 is an improved mixture of three extracts (Psidium guajava, Camellia sinensis, and Rosa hybrida) recognized by the Ministry of Food and Drug Safety of Korea as a health food ingredient that alleviates allergic rhinitis. The current animal efficacy study was performed to assess its probability of improving COVID-19 symptoms. BEN815 treatment significantly increased the survival of K18-hACE2 transgenic mice and reduced viral titers in the lungs at 5 days post infection (DPI). Furthermore, the lungs of the treated mice showed mild tissue damage at 5 DPI and nearly complete recovery from COVID-19 at 14 DPI. BEN815 appears to be an effective and minimally toxic anti-SARS-CoV-2 agent in mice and has potential for clinical applications.

Pathological assessment and tissue tropism of two different Egyptian infectious bronchitis strains.

Avian infectious bronchitis is one of the most common viral infections in chickens affecting all ages. The tropism of infectious bronchitis virus (IBV) strains became broader and more variable posing major implications for the effective control of IBV infection. In this study, two IBV viruses representing classic and variant strains were inoculated intranasally into day-old SPF chicks (105 EID50/0.2ml/bird). Clinical signs were observed for 15 days post-infection (DPI). Five chicks from each group were euthanized at 2, 4, 6, 8, 10, 12, and 15 DPI for histopathology and virus antigen detection by IHC and quantitative rRT-PCR. Results revealed that both classic and variant IBV strains induced mild clinical signs with no mortalities and fewer various histopathological lesions in infected SPF chickens. Although the viruses were detected by rRT-PCR up to 12DPI, the affected tissues showed regeneration after 10DPI with IHC revealing no IBV antigen. In summary, no differences were found in the behaviour of both IBV isolates in chickens. The broad tissue tropism for both IBV strains as indicated by viral antigen detection in various organs with no clinical or gross lesion suggest that the main cause of death in IBV infection under field conditions occurs as a result of complication with secondary infections rather single IBV infection. Due to positive immunostaining in the bursa, it is thought that IBV infection has immunosuppressive consequences, hence further study is required to validate this impact.

Oil Based Inactivated Vaccine Formulation for Furunculosis (A. salmonicida) and Protective Immune Response of Rainbow Trout and Brown Trout

The development and growth of the fisheries and aquaculture industries are significantly hampered by illnesses. It is critical to combat pathogenic illnesses, especially bacterial ones. Furunculosis in salmon is mostly brought on by Aeromonas salmonicida in rainbow and brown trout. To control this pathogen, vaccines have been identified as a significant tool. In the present study, we have formulated an inactivated vaccine with oil as an adjuvant and estimated its efficacy. The lethal dose of ArS-Pak-19, was calculated and injected intraperitoneally to the fishes. To analyze the infection, samples of kidney, liver, spleen, and blood were collected at specific times. To estimate the immunogenicity of the vaccine, an experiment was designed. One hundred sixty fishes were distributed into 8 tanks including, six experimental groups and two control groups with its replicates, vaccines injected intraperitoneally 1.6 × 107, 1.6 × 108, and 1.6 × 109 and blood samples were taken fortnightly for 56 days to calculate the antibodies titers. After immunization these groups were challenged with Aeromonas salmonicida (ArS-Pak-19) intraperitoneally. At 7th day of post infection, it appeared in the liver, spleen, and kidney. The relative percentage of survival was estimated with control groups at 30 days after challenge. The relative percentage of survival was 80%. The IgM titers were higher at 24 days of post immunization. We also analyzed that antibodies non-specifically bound with the A-layer of Aeromonas salmonicida. The findings of this study offer evidence that vaccinations boost fishes immunity and serve as a roadmap to further vaccination initiatives.

Dynamics of Plasmodium berghei NK-65 parasitaemia and CD3+CD4+CD25+Fox-p3+ T-regulatory cells in experimentally induced malaria during early, mid, and late-pregnancy in BALB/c mice.

Malaria in pregnancy causes a dual brunt on the mother as well as the foetus. Upregulation of T-regulatory cells (Tregs) during pregnancy allows tolerance towards the growing foetus, their suppression predisposes the mother to infections. This study analyzed the levels of CD3+CD4+CD25+Fox-p3+ Tregs, parasitaemia, maternal and foetal outcomes in BALB/c mice infected with P. berghei NK65 during early-, mid-, and late-pregnancy. Total of 114 mice, non-pregnant non-infected (n = 6), non-pregnant infected (n = 12), pregnant non-infected (n = 48) and pregnant infected (n = 48) were included in the study. Infected groups were inoculated intra-peritoneally with 1 × 106 P. berghei infected RBCs during early-, mid-, and late- pregnancy (D6, D10, and D14 respectively). Six mice from each stage were sacrificed on the 5th and 7th day post-infection (DPI) to evaluate parasitaemia (staining) and Tregs from splenocytes (by flow cytometry). The parasitaemia was significantly higher among early pregnancy infected mice (≥ 70%) than mid-pregnancy infected (40-70%), late pregnancy infected (50-65%), and non-pregnant infected mice (≤ 50%) (p < 0.05). The level of Tregs was significantly higher among non-pregnant infected mice as compared to non-pregnant non-infected mice (%Tregs 0.86 vs. 0.44). Among pregnant mice, the levels of Tregs in infected mice were lower than in non-infected mice during all stages of pregnancy. None of the mice infected during early- and mid-pregnancy survived at 6DPI and 7DPI, respectively, and those infected during late-pregnancy delivered premature pups. In contrast to non-pregnant mice, the levels of Tregs among pregnant mice decrease when malaria infection is acquired thereby leading to adverse pregnancy outcomes. The online version contains supplementary material available at 10.1007/s12088-023-01089-2.

Coccidiosis infection and growth performance of broilers in experimental trials: insights from a meta-analysis including modulating factors

An infection by protozoa Eimeria spp. can cause coccidiosis, which negatively affects broiler chicken performance and causes economic and production losses. To understand the effect of coccidiosis on broilers' performance, we evaluated the independent variables and their interactions on the severity of coccidiosis in broilers that cause variation (Δ) of average daily feed intake (ADFI), average daily gain (ADG), and gain per feed (G:F) of broiler chicks using a meta-analysis approach. A database of 55 papers describing 63 experiments was gathered; broilers were challenged by Eimeria species (E. acervulina, E. maxima, E. tenella, and mixed) and at least 2 variables among ADFI, ADG, and feed conversion ratio (FCR) were studied. The variation induced by the challenge was calculated relative to the control group of each experiment. The indirect factors evaluated were days postinfection (DPI), Eimeria type and dose, infection age (IA), bird's mean age in the analyzed period, genetic line, sex, and whether they were raised in a cage or a pen. Graphical, correlation, and variance analyses were performed to evaluate the form of the responses. Then, a linear plateau model was adjusted for each response variable as a function of DPI to determine the consequences of the disease on the variation of performance over time after infection. The impact of the infection challenge on the variation of performance vs. nonchallenge broilers was only impacted by DPI (P < 0.05). The adjustment of the data with the linear plateau model allows us to determine the host response to the coccidiosis disease at different stages. At 5 DPI (acute phase), ΔADFI, ΔADG, ΔG:F were of -19.0; -39.8, and -25.5, respectively. After almost 13 DPI birds achieved the recovery phase for all variables with Δ varying from -19 to -3.75% for ADFI, from -39.8 to -10.5% for ADG, and from -25.5 to -7.24% for G:F. The Eimeria impact was higher in ADG than ADFI in all periods due to Eimeria aggressive action form causing lesions in gut epithelial reducing the use of nutrients and energy. The results can be used as a quantitative approach to determine the consequences of Eimeria spp. on broiler performance.

In ovo administration of a phage cocktail partially prevents colibacillosis in chicks

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the main bacterial disease in poultry leading to significant economic losses worldwide. Antibiotic treatments favor the emergence of multidrug-resistant bacteria, and preventive measures are insufficient to control the disease. There is increasing interest in using the potential of bacteriophages, not only for phage therapy but also for prevention and biocontrol. This study aimed to evaluate the efficacy of a phage cocktail administered in ovo to prevent avian colibacillosis in chicks. When 4 different phages (REC, ESCO3, ESCO47, and ESCO58), stable under avian physiological conditions, were combined and inoculated at 17 embryogenic days (ED), they were transmitted to the newly hatched chicks. In a second trial, the 4-phage cocktail was inoculated into the allantoic fluid at ED16 and after hatch 1-day-old chicks were challenged with the O2 APEC strain BEN4358 inoculated subcutaneously. Two phages (REC and ESCO3) were still detected in the ceca of surviving chicks at the end of the experiment (7-days postinfection). Chicks that received the phages in ovo did not develop colibacillosis lesions and showed a significant decrease in intestinal BEN4358 load (8.00×107 CFU/g) compared to the challenged chicks (4.52×108 CFU/g). The majority of the reisolated bacteria from the ceca of surviving chicks had developed full resistance to ESCO3 phage, and only 3 were resistant to REC phage. The partially or complete resistance of REC phage induced a considerable cost to bacterial virulence. Here, we showed that phages inoculated in ovo can partially prevent colibacillosis in 1-wk-old chicks. The reduction in the APEC load in the gut and the decreased virulence of some resistant isolates could also contribute to control the disease.

Dynamic RNA profiles in the small intestinal epithelia of cats after Toxoplasma gondii infection

BackgroundFelids are the only definitive hosts of Toxoplasma gondii. However, the biological features of the feline small intestine following T. gondii infection are poorly understood. We investigated the changes in the expression of RNAs (including mRNAs, long non-coding RNAs and circular RNAs) in the small intestinal epithelia of cats following T. gondii infection to improve our understanding of the life cycle of T. gondii and cat responses to T. gondii infection.MethodsFifteen cats were randomly assigned to five groups, and the infection groups were inoculated with 600 tissue cysts of the T. gondii Pru strain by gavage. The small intestinal epithelia of cats were collected at 6, 10, 14, and 30 days post infection (DPI). Using high-throughput RNA sequencing (RNA-seq), we investigated the changes in RNA expression. The expression levels of differentially expressed (DE) genes and non-coding RNAs (ncRNAs) identified by RNA-seq were validated by quantitative reverse transcription PCR (qRT-PCR). Differential expression was determined using the DESeq R package.ResultsIn total, 207 annotated lncRNAs, 20,552 novel lncRNAs, 3342 novel circRNAs and 19,409 mRNAs were identified. Among these, 70 to 344 DE mRNAs, lncRNAs and circRNAs were detected, and the post-cleavage binding sites between 725 ncRNAs and 2082 miRNAs were predicted. Using the co-location method, we predicted that a total of 235 lncRNAs target 1044 protein-coding genes, while the results of co-expression analysis revealed that 174 lncRNAs target 2097 mRNAs. Pathway enrichment analyses of the genes targeted by ncRNAs suggested that most ncRNAs were significantly enriched in immune or diseases-related pathways. NcRNA regulatory networks revealed that a single ncRNA could be directly or indirectly regulated by multiple genes or ncRNAs that could influence the immune response of cats. Co-expression analysis showed that 242 circRNAs, mainly involved in immune responses, were significantly associated with T. gondii infection. In contrast, 1352 protein coding RNAs, mainly involved in nucleic acid process/repair pathways or oocyte development pathways, were negatively associated with T. gondii infection.ConclusionsThis study is the first to reveal the expression profiles of circRNAs, lncRNAs and mRNAs in the cat small intestine following T. gondii infection and will facilitate the elucidation of the role of ncRNAs in the pathogenesis of T. gondii infection in its definitive host, thereby facilitating the development of novel intervention strategies against T. gondii infection in humans and animals.Graphical

Development of a new immunodiagnostic tool for poultry coccidiosis using an antigen-capture sandwich assay based on monoclonal antibodies detecting an immunodominant antigen of Eimeria

This study was conducted to develop an antigen-capture ELISA that detects an immunodominant antigen of Eimeria, 3-1E which is present in all Eimeria species, using a set of 3-1E-specific mouse monoclonal antibodies (mAbs). Highly sensitive 3-1E-specific antigen-capture ELISA was established using compatible mAb pairs (#318 and #320) selected from 6 mAbs (#312, #317, #318, #319, #320, and #323) with high binding activity against recombinant 3-1E protein. These anti-3-1E mAbs specifically recognized E. tenella sporozoites and a higher level of 3-1E was detected in the lysate of sporozoites than in sporocysts. Immunofluorescence assay (IFA) using 2 mAbs (#318 and #320) showed specific staining around the membrane of E. tenella sporozoites. In order to measure the changes in the 3-1E level during in coccidiosis, serum, feces, jejunal, and cecal contents were individually collected daily for 7-days postinfection (dpi) with E. maxima and E. tenella. The new ELISA was sensitive and specific for 3-1E detection in all samples collected daily from E. maxima- and E. tenella-infected chickens for a week, and the detection sensitivity ranges were 2 to 5 ng/mL and 1 to 5 ng/mL in serum, 4 to 25 ng/mL and 4 to 30 ng/mL in feces, 1 to 3 ng/mL and 1 to 10 ng/mL in cecal contents, and 3 to 65 ng/mL and 4 to 22 ng/mL in jejunal contents. Following coccidiosis, the overall 3-1E levels started to increase from 4 dpi, and the highest production was shown on 5 dpi. Among the samples collected from Eimeria-infected chickens, the highest detection level was found in the jejunal contents of E. maxima-infected chickens. Furthermore, the level of IFN-γ in serum was significantly (P < 0.05) increased from 3 dpi and peaked on 5 dpi post E. maxima infection. Post E. tenella infection, the level of IFN-γ in serum gradually (P < 0.05) increased from 2 to 5 dpi and plateaued at 7 dpi. The level of TNF-α in serum was rapidly (P < 0.05) increased from 4 dpi and those levels were kept until 7 dpi post both Eimeria infections (E. maxima and E. tenella). More importantly, the daily changes in the 3-1E levels in different samples from E. maxima- and E. tenella-infected chickens were effectively monitored with this new antigen-capture ELISA. Therefore, this new immunoassay is a sensitive diagnostic tool to monitor coccidiosis in a large field population in the commercial poultry farms before clinical symptoms develop using serum, feces, and gut samples during the entire period of infection cycle starting from 1 d after infection.

Rat lungworm (Angiostrongylus cantonensis) active larval emergence from deceased bubble pond snails (Bullastra lessoni) into water.

Angiostrongylus cantonensis (the rat lungworm) is a zoonotic parasite of non-permissive accidental (dogs, humans, horses, marsupials, birds) hosts. The 3rd stage larvae (L3s) in the intermediate host (molluscs) act as the source of infection for accidental hosts through ingestion. Larvae can spontaneously emerge from dead gastropods (slugs and snails) in water, which are experimentally infective to rats. We sought to identify the time when infective A. cantonensis larvae can autonomously leave dead experimentally infected Bullastra lessoni snails. The proportion of A. cantonensis larvae that emerge from crushed and submerged B. lessoni is higher in snails 62 days post-infection (DPI) (30.3%). The total larval burden of snails increases at 91 DPI, indicating that emerged larvae subsequently get recycled by the population. There appears to be a window of opportunity between 1 and 3 months for infective larvae to autonomously escape dead snails. From a human and veterinary medicine viewpoint, the mode of infection needs to be considered; whether that be through ingestion of an infected gastropod, or via drinking water contaminated with escaped larvae.

Histopathology and virulence of an in vitro-adapted Trypanosoma evansi TEDC 953 strain (Thailand isolate) in mice.

Trypanosoma evansi is a blood and tissue protozoan parasite affecting domestic and wild animals. The T. evansi Thai strain, namely, T. evansi from dairy cattle number 953 (TEDC 953) strain, has been successfully isolated from dairy cattle and cultivated in vitro. The in vitro-cultivated parasite is useful for biological studies, evaluation of novel chemotherapeutic agents, and production of antigens for diagnostic tests. This study aimed to observe the histopathology and virulence of an in vitro-adapted T. evansi TEDC 953 strain in vivo. The histopathology and virulence of the TEDC 953 strain were clarified in mice. Six mice were infected with 1 × 105 trypomastigotes of TEDC 953 strain intraperitoneally, and four mice were in the negative control. Parasitemia was monitored daily, and the mice were euthanized on 30 days post-infection (DPI). Internal organs were collected for histopathological examination using hematoxylin and eosin staining. Histopathological lesions were found in the liver, lung, heart, kidney, spleen, and brain of the inoculated mice. The main histopathological feature was lymphoplasmacytic inflammation in parenchyma and perivascular areas of multiple organs, and the severity of histopathological changes was related to the presence of trypomastigotes in the regional vessels. Granulomatous inflammation was seen in meninges, pleura, renal capsule, renal pelvis, and spleen of some infected mice. Four mice died at 17, 24, 26, and 27 DPI with an average parasitemia of 4.05 × 1011 trypomastigotes/mL. The average survival time was 23.5 DPI (mice = 4). This study confirmed that the TEDC 953 strain is infectious and pathogenic in mice after the continuously cultivated in vitro. To replace the use of experimental animals, the in vitro-cultivated parasite can be used instead in further studies.

Inducible nitric oxide synthase deficiency promotes murine-β-coronavirus induced demyelination

BackgroundMultiple sclerosis (MS) is characterized by neuroinflammation and demyelination orchestrated by activated neuroglial cells, CNS infiltrating leukocytes, and their reciprocal interactions through inflammatory signals. An inflammatory stimulus triggers inducible nitric oxide synthase (NOS2), a pro-inflammatory marker of microglia/macrophages (MG/Mφ) to catalyze sustained nitric oxide production. NOS2 during neuroinflammation, has been associated with MS disease pathology; however, studies dissecting its role in demyelination are limited. We studied the role of NOS2 in a recombinant β-coronavirus-MHV-RSA59 induced neuroinflammation, an experimental animal model mimicking the pathological hallmarks of MS: neuroinflammatory demyelination and axonal degeneration.ObjectiveUnderstanding the role of NOS2 in murine-β-coronavirus-MHV-RSA59 demyelination.MethodsBrain and spinal cords from mock and RSA59 infected 4–5-week-old MHV-free C57BL/6 mice (WT) and NOS2-/- mice were harvested at different disease phases post infection (p.i.) (day 5/6-acute, day 9/10-acute-adaptive and day 30-chronic phase) and compared for pathological outcomes.ResultsNOS2 was upregulated at the acute phase of RSA59-induced disease in WT mice and its deficiency resulted in severe disease and reduced survival at the acute-adaptive transition phase. Low survival in NOS2-/- mice was attributed to (i) high neuroinflammation resulting from increased accumulation of macrophages and neutrophils and (ii) Iba1 + phagocytic MG/Mφ mediated-early demyelination as observed at this phase. The phagocytic phenotype of CNS MG/Mφ was confirmed by significantly higher mRNA transcripts of phagocyte markers-CD206, TREM2, and Arg1 and double immunolabelling of Iba1 with MBP and PLP. Further, NOS2 deficiency led to exacerbated demyelination at the chronic phase as well.ConclusionTaken together the results imply that the immune system failed to control the disease progression in the absence of NOS2. Thus, our observations highlight a protective role of NOS2 in murine-β-coronavirus induced demyelination.

A178 ELUCIDATING THE ROLE OF THE LEUCINE-RICH REPEAT KINASE 2 G2019S MUTATION IN CROHN’S DISEASE PATHOGENESIS USING A CITROBACTER RODENTIUM INFECTIOUS COLITIS MODEL

Abstract Background Associations have been found linking certain LRRK2 kinase domain gain-of-function variants, such as G2019S, to the development of Crohn’s disease and Parkinson’s disease, yet their exact roles in pathogenesis remains elusive. LRRK2 is most robustly expressed in circulating and tissue-resident immune cells, such as neutrophils, lymphocytes, and macrophages. Myeloid cells deficient in LRRK2 exhibit defective antimicrobial responses, such as reduced production of reactive oxygen species in response to microbial stimuli and reduced bactericidal activity in response to infection. As an enteric colitis-inducing extracellular pathogen, Citrobacter rodentium can help us better understand the consequences of LRRK2 kinase hyperactivity on intestinal inflammation by correlating pathogen burden with key host response parameters over the course of infection. Purpose To investigate the effects of the Crohn’s and Parkinson’s disease-associated Lrrk2 G2019S hyper-kinase mutation on pathogen burden and colonic inflammation in the context of C. rodentium-induced infectious colitis. Method Wild-type and Lrrk2 G2019S mutant mice (7-8 weeks old) were fasted for 4 hours then infected with 1 x 108 CFU of C. rodentium in a 3% NaHCO3 solution by oral gavage. Body weight, faecal pathogen burden, and faecal lipocalin-2 (Lcn2) concentrations were measured at 2, 4, 7, 9, 10, 12, and 14 days post-infection (DPI). Systemic pathogen burden (as measured in the mesenteric lymph nodes and spleen), colon length, colonic inflammatory gene expression, and histopathological scoring were assessed at 7, 10, and 14 DPI. Result(s) While G2019S mice exhibited marginally higher C. rodentium loads at certain timepoints, no significant differences were found in overall pathogen burden or pathogen clearance rates between genotypes over the first 14 days of infection. Faecal pathogen load peaked at 7-9 DPI in both WT and G2019S mice, which correlated with detectable levels of C. rodentium in the mesenteric lymph nodes and spleens of some mice at 7 DPI. Lcn2 secretion and the expression of inflammatory and antimicrobial genes of interest were induced robustly over the course of infection. They peaked and ebbed at timepoints correlating well with pathogen burden; however, no significant differences were observed between WT and G2019S mutant mice at the various timepoints assessed. Conclusion(s) Mice expressing the G2019S Lrrk2 mutation exhibit neither defective pathogen control nor deleterious hyperinflammation – compared to WT mice – when infected with C. rodentium. Further research will aim to investigate the role of this Lrrk2 variant in additional models of intestinal inflammation. Please acknowledge all funding agencies by checking the applicable boxes below CCC, CIHR Disclosure of Interest None Declared

Pathogenesis of Kasokero virus in experimentally infected Egyptian rousette bats (Rousettus aegyptiacus)

Egyptian rousette bats (ERBs; Rousettus aegyptiacus; family Pteropodidae) are associated with a growing number of bunyaviruses of public health importance, including Kasokero virus (KASV), which was first identified as a zoonosis in Uganda in 1977. In this study, formalin-fixed paraffin-embedded tissues from a previous experiment in which KASV infection was confirmed in 18 experimentally infected ERBs were used for an in-depth analysis using histopathology, in situ hybridization (ISH) for detection of viral RNA, immunohistochemistry (IHC) to assess the mononuclear phagocyte system response, and quantitative digital image analysis to investigate virus clearance from the liver and spleen within a spatial context. Significant gross and histological lesions were limited to the liver, where KASV-infected bats developed mild to moderate, acute viral hepatitis, which was first observed at 3 days postinfection (DPI), peaked at 6 DPI, and was resolved by 20 DPI. A subset of bats had glycogen depletion (n = 10) and hepatic necrosis (n = 3), rarely with intralesional bacteria (n = 1). Virus replication was confirmed by ISH in the liver, spleen, lymph nodes, and tongue. In the liver, KASV replicated in the cytoplasm of hepatocytes, to a lesser extent in mononuclear phagocytes, and rarely in presumptive endothelial cells. Most KASV RNA, as detected by ISH, was cleared from the spleen and liver by 6 DPI. It is concluded that ERBs have effective mechanisms to respond to this virus, clearing it without evidence of clinical disease.

Experimental Infection of Domestic Pigs (Sus scrofa) with Rift Valley Fever Virus.

Rift valley fever (RVF), caused by the RVF virus (RVFV), is a vector-borne zoonotic disease that primarily affects domestic ruminants. Abortion storms and neonatal deaths characterise the disease in animals. Humans develop flu-like symptoms, which can progress to severe disease. The susceptibility of domestic pigs (Sus scrofa domesticus) to RVFV remains unresolved due to conflicting experimental infection results. To address this, we infected two groups of pregnant sows, neonates and weaners, each with a different RVFV isolate, and a third group of weaners with a mixture of the two viruses. Serum, blood and oral, nasal and rectal swabs were collected periodically, and two neonates and a weaner from group 1 and 2 euthanised from 2 days post infection (DPI), with necropsy and histopathology specimens collected. Sera and organ pools, blood and oronasorectal swabs were tested for RVFV antibodies and RNA. Results confirmed that pigs can be experimentally infected with RVFV, although subclinically, and that pregnant sows can abort following infection. Presence of viral RNA in oronasorectal swab pools on 28 DPI suggest that pigs may shed RVFV for at least one month. It is concluded that precautions should be applied when handling pig body fluids and carcasses during RVF outbreaks.

Pathogenicity and pathogenesis of a recent highly pathogenic avian influenza subtype H5N8 in mule ducklings in Egypt.

In late 2017, an H5N8 highly pathogenic avian influenza (HPAI) virus, clade 2.3.4.4, was isolated from domestic ducks in Egypt, which was associated with high morbidity and low mortality. The pathogenicity increased due to the continuous circulation of virus in ducks. Thus, this study aimed to monitor the pathogenesis and pathogenicity of new H5N8 Avian influenza (AI) virus in mule ducklings. The lethal dose 50 (LD50) for this new local HPAI H5N8 isolate was calculated. Twenty ducklings were inoculated with 0.1 mL of dilution containing 10 LD50 HPAI per duck. The clinical signs and mortalities were recorded until 30 days post-infection (DPI) to confirm viral pathogenesis. Reverse transcription polymerase chain reaction was used to detect viral shedding from collected cloacal swabs after 3rd, 5th, 7th, 10th, 14th, 21st, and 30th DPI. The main histopathological lesions associated with the presence of HPAI virus were also recorded on the 3rd and 14th DPI. The result showed that the LD50 of the new HPAI H5N8 was 104 log10. Clinical signs were observed after 2nd DPI, but it was clinically severe on 3rd, 4th, and 5th DPI in the form of respiratory and gastric disorders, forming 90% of all diseased ducklings, whereas 30% of the infected ducks only showed nervous signs. The mortality rate peaked on 4th and 5th DPI with a cumulative mortality rate of 60% for the inoculated ducks, whereas no mortality was recorded after 6th DPI. Dead ducks showed typical postmortem lesions of AI disease. Necrosis and ecchymotic or petechial hemorrhages on the heart, pancreas, liver, and spleen were observed, whereas the lung showed pneumonia. With regard to viral shedding, infected ducklings shed the virus from its gut until 7th DPI, but the number of duck shedders gradually decreased until 14th DPI after viral shedding. The histopathological findings indicated that the spleen and thymus showed necrosis and hemorrhages, whereas the brain showed multifocal malacic foci and spread meningitis. Moreover, the lung had intrabronchial hyaline degeneration and fibrinous pneumonia on 3rd DPI. Furthermore, the liver showed multifocal necrotic foci and subcapsular hemorrhage, whereas the kidney showed remarkable tubular degeneration, mostly within the collecting tubules. Furthermore, the heart showed marked myocardiolysis of the cardiac muscle fibers. On 14th DPI, all histopathological lesions of the examined organs were restored to normal. The currently circulating HPAI H5N8 virus strain has high virulence, particularly for imported mule ducks that originated from non-vaccinated breeder ducks. Therefore, vaccination and quarantine measures must be applied on imported 1-day-old mule ducklings. Moreover, the pathogenesis must be reviewed and monitored for updating circulating AI strains caused by the continuous and rapid evolution of AI viruses.

Korean Red Ginseng Relieves Inflammation and Modulates Immune Response Induced by Pseudo-Type SARS-CoV-2.

Few studies have reported the therapeutic effects of Korean red ginseng (KRG) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the positive effects of KRG on other viruses have been reported and the effects of KRG on pulmonary inflammatory diseases have also been studied. Therefore, this study investigated the therapeutic effects of KRG-water extract (KRG-WE) in a pseudo-type SARS-CoV-2 (PSV)-induced lung injury model. Constructing the pseudovirus, human angiotensin-converting enzyme 2 (hACE2) transgenic mice were infected via intranasal injection that had been orally administered with KRG-WE for six weeks. After 7-days post infection (dpi), the antiviral effects of KRG-WE were confirmed, followed by real-time polymerase chain reaction (PCR), western blot analysis, flow cytometric analysis, and an enzyme-linked immunoassay (ELISA). KRG-WE significantly inhibited an increase in immunoglobulin caused by PSV. Furthermore, KRG-WE effectively suppressed alveolar macrophages (AMs) inside the lungs and helped normalize the population of other immune cells. In addition, virus-induced gene expression and inflammatory signals such as nuclear factor-kappa B and other upstream molecules were downregulated. Moreover, KRG-WE also normalized gene expression and protein activity in the spleen. In conclusion, KRG-WE reduced AMs, normalized the immune response, and decreased the expression of inflammatory genes and activation of signaling pathway phosphorylation, thereby exhibiting anti-inflammatory effects and attenuating lung damage.

Vector Competence of a Coastal Population of Aedes aegypti for Dengue 2 and 3 Virus Serotypes in Kenya

Aedes aegypti is the primary vector of dengue, an arboviral disease caused by dengue virus (DENV) that exists as four distinct serotypes (DENV 1‐4). While all four DENV serotypes circulate in Kenya, differential distribution of the serotypes in specific regions suggests virus transmission may differ among local vector populations. In this study, we tested the hypothesis that a coastal Ae. aegypti population (Rabai, Kilifi County) varies in its ability to transmit DENV‐2 (predominant) and DENV‐3 (less dominant) and that transmission is related to Ae. aegypti subspecies—domestic Ae. aegypti aegypti (Aaa) and sylvtic Ae. aegypti formosus (Aaf). We orally exposed F1 females (3‐10 days old) to blood meals containing DENV‐2 (10 5.30 pfu/ml) or DENV‐3 (10 5.13 pfu/ml), tested them individually for infection (body), dissemination (legs), and transmission (saliva) at 7, 14, and 21 days postinfection (DPI), respectively, and compared the rates between the serotypes. We analyzed cytochrome c oxidase I gene (cox-I) sequences among DENV‐susceptible and nonsusceptible cohorts. Of 489 mosquitoes tested (DENV‐2: 240; DENV‐3: 249), we found consistently higher but nonsignificant rates of infection (16% vs. 10%), dissemination (47% (18/38) vs. 35% (9/26)), and transmission (39% (7/18) vs. 11% (1/9)) for DENV‐2 than DENV‐3. However, DENV‐2 exhibited a shorter extrinsic incubation period (EIP) for disseminated infection (7‐DPI vs. 14‐DPI) and transmission (14‐DPI vs. 21‐DPI) compared to DENV‐3. Two cox-I lineages were recovered in phylogeny, one predominantly clustered with referenced Aaa and a minor lineage grouped with Aaf. Infected mosquitoes and those with disseminated infection were represented in both lineages; those that transmitted the viruses grouped with the Aaa‐associated lineage only. We conclude that the coastal Ae. aegypti population is a competent vector for DENV‐2 and DENV‐3 likely driven by the domestic Aaa that is predominant. The shorter EIP to attain dissemination and transmission for DENV‐2 could favour its transmission over DENV‐3.