Inflammation, the kynurenines, and mucosal injury during human experimental enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in children and travelers, especially in low- and middle-income countries. ETEC is a non-invasive gut pathogen colonizing the small intestinal wall before secreting diarrhea-inducing enterotoxins. We sought to investigate the impact of ETEC infection on local and systemic host defenses by examining plasma markers of inflammation and mucosal injury as well as kynurenine pathway metabolites. Plasma samples from 21 volunteers experimentally infected with ETEC were collected before and 1, 2, 3, and 7 days after ingesting the ETEC dose, and grouped based on the level of intestinal ETEC proliferation: 14 volunteers experienced substantial proliferation (SP) and 7 had low proliferation (LP). Plasma markers of inflammation, kynurenine pathway metabolites, and related cofactors (vitamins B2 and B6) were quantified using targeted mass spectrometry, whereas ELISA was used to quantify the mucosal injury markers, regenerating islet-derived protein 3A (Reg3a), and intestinal fatty acid-binding protein 2 (iFABP). We observed increased concentrations of plasma C-reactive protein (CRP), serum amyloid A (SAA), neopterin, kynurenine/tryptophan ratio (KTR), and Reg3a in the SP group following dose ingestion. Vitamin B6 forms, pyridoxal 5'-phosphate and pyridoxal, decreased over time in the SP group. CRP, SAA, and pyridoxic acid ratio correlated with ETEC proliferation levels. The changes following experimental ETEC infection indicate that ETEC, despite causing a non-invasive infection, induces systemic inflammation and mucosal injury when proliferating substantially, even in cases without diarrhea. It is conceivable that ETEC infections, especially when repeated, contribute to negative health impacts on children in ETEC endemic areas.

Host specificity and virulence of Flavobacterium psychrophilum: a comparative study in ayu (Plecoglossus altivelis) and rainbow trout (Oncorhynchus mykiss) hosts.

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease, is a devastating, worldwide distributed, fish pathogen causing significant economic loss in inland fish farms. Previous epidemiological studies showed that prevalent clonal complexes (CC) differ in fish species affected with disease such as rainbow trout, coho salmon and ayu, indicating significant associations between particular F. psychrophilum genotypes and host species. Yet, whether the population structure is driven by the trade of fish and eggs or by host-specific pathogenicity is uncertain. Notably, all F. psychrophilum isolates retrieved from ayu belong to Type-3 O antigen (O-Ag) whereas only very few strains retrieved from other fish species possess this O-Ag, suggesting a role in outbreaks affecting ayu. Thus, we investigated the links between genotype and pathogenicity by conducting comparative bath infection challenges in two fish hosts, ayu and rainbow trout, for a collection of isolates representing different MLST genotypes and O-Ag. Highly virulent strains in one host species exhibited low to no virulence in the other. F. psychrophilum strains associated with ayu and possessing Type-3 O-Ag demonstrated significant variability in pathogenicity in ayu, ranging from avirulent to highly virulent. Strikingly, F. psychrophilum strains retrieved from rainbow trout and possessing the Type-3 O-Ag were virulent for rainbow trout but not for ayu, indicating that Type-3 O-Ag alone is not sufficient for pathogenicity in ayu, nor does it prevent pathogenicity in rainbow trout. This study revealed that the association between a particular CC and host species partly depends on the pathogen's adaptation to specific host species.

Genetic diversity of Cryptosporidium spp., Encephalitozoon spp. and Enterocytozoon bieneusi in feral and captive pigeons in Central Europe.

Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon spp. are the most common protistan parasites of vertebrates. The results show that pigeon populations in Central Europe are parasitised by different species of Cryptosporidium and genotypes of microsporidia of the genera Enterocytozoon and Encephalitozoon. A total of 634 and 306 faecal samples of captive and feral pigeons (Columba livia f. domestica) from 44 locations in the Czech Republic, Slovakia and Poland were analysed for the presence of parasites by microscopy and PCR/sequence analysis of small subunit ribosomal RNA (18S rDNA), 60 kDa glycoprotein (gp60) and internal transcribed spacer (ITS) of SSU rDNA. Phylogenetic analyses revealed the presence of C. meleagridis, C. baileyi, C. parvum, C. andersoni, C. muris, C. galli and C. ornithophilus, E. hellem genotype 1A and 2B, E. cuniculi genotype I and II and E. bieneusi genotype Peru 6, CHN-F1, D, Peru 8, Type IV, ZY37, E, CHN4, SCF2 and WR4. Captive pigeons were significantly more frequently parasitised with screened parasite than feral pigeons. Cryptosporidium meleagridis IIIa and a new subtype IIIl have been described, the oocysts of which are not infectious to immunodeficient mice, whereas chickens are susceptible. This investigation demonstrates that pigeons can be hosts to numerous species, genotypes and subtypes of the studied parasites. Consequently, they represent a potential source of infection for both livestock and humans.

Early stage Fasciola hepatica infection in growing cattle: impact on weight gain and liver weight determined in flukicide efficacy studies.

Although the negative impact of liver fluke (Fasciola hepatica) infection on production and health in cattle is generally accepted, results of individual research have been variable, ranging from important negative impacts on the animal to minimal or no impact. To add information on the impact of F. hepatica infection in growing cattle, weight gain and liver weight of young experimentally infected animals from seven controlled efficacy studies were analyzed. In each study, fluke naïve animals were inoculated with approximately 450 to 500 F. hepatica encysted metacercariae, blocked on body weight and randomly assigned into one untreated group (controls) and groups which were administered an experimental flukicide when the flukes were 4 weeks old (migrating) and sacrificed 8 weeks thereafter (12 weeks after inoculation). Data of groups which demonstrated >90% reduction of fluke counts following treatment and groups left untreated (total 103 and 47 animals, respectively) were compared. There was a significant (p < 0.0001) negative association between fluke count and weight gain while fluke count and liver weight and fluke count and relative liver weight were positively associated (p < 0.0001). Over the 8-week post-treatment period, flukicide-treated cattle had almost 15% more weight gain than the controls (50.9 kg vs. 44.4 kg; p = 0.0003). Absolute and relative liver weight was significantly (p < 0.0001) lower in flukicide-treated compared to untreated cattle. Overall, this analysis provided evidence of a substantial negative effect of early (migrating) liver fluke infection on the growth of young cattle, likely due to pathology of the liver and associated reduction in its function as the central organ for bioenergy and protein metabolism.

Deciphering host immunity to malaria using systems immunology.

A century of conceptual and technological advances in infectious disease research has changed the face of medicine. However, there remains a lack of effective interventions and a poor understanding of host immunity to the most significant and complex pathogens, including malaria. The development of successful interventions against such intractable diseases requires a comprehensive understanding of host-pathogen immune responses. A major advance of the past decade has been a paradigm switch in thinking from the contemporary reductionist (gene-by-gene or protein-by-protein) view to a more holistic (whole organism) view. Also, a recognition that host-pathogen immunity is composed of complex, dynamic interactions of cellular and molecular components and networks that cannot be represented by any individual component in isolation. Systems immunology integrates the field of immunology with omics technologies and computational sciences to comprehensively interrogate the immune response at a systems level. Herein, we describe the system immunology toolkit and report recent studies deploying systems-level approaches in the context of natural exposure to malaria or controlled human malaria infection. We contribute our perspective on the potential of systems immunity for the rational design and development of effective interventions to improve global public health.

Experimental Infection of North American Deer Mice with Clade I and II Monkeypox Virus Isolates.

The global spread of monkeypox virus has raised concerns over the establishment of novel enzootic reservoirs in expanded geographic regions. We demonstrate that although deer mice are permissive to experimental infection with clade I and II monkeypox viruses, the infection is short-lived and has limited capability for active transmission.

A review of reptile virus experimental infection studies.

Despite recent advances in molecular techniques, infection studies remain an important tool for biosecurity, veterinary and conservation medicines. Experimental infection studies are performed for many reasons: to investigate causal links between pathogens and disease, to study host species susceptibility, to study immune response to inoculation, to investigate pathogen transmission and to investigate methods for infection control. Experimental infection studies using viruses in reptiles have been conducted sporadically since at least the 1930s and this continues to be a fertile area of research. This review catalogues previously published research in the field. The key parameters of each study are tabulated, providing a summary of more than 100 experiments linked to their original publications. Common themes and trends within the data are discussed.

Quantitative analysis of viremia and viral shedding in pigs infected experimentally with classical swine fever virus isolates obtained from recent outbreaks in Japan.

Although classical swine fever occurred in September 2018 for the first time in 26 years, its virulence is thought to be moderate based on field observations by veterinary authorities and our previous experimental infections. We quantified viremia and viral shedding in pigs infected with recent Japanese classical swine fever virus isolates, as well as a highly virulent strain. The results show that pigs infected with the Japanese strains exhibited lower viremia and viral shedding than those infected with the highly virulent strain. However, horizontal transmission occurred in pigs infected with the Japanese strains, similar to those infected with the highly virulent strain. Additionally, viremia and neuralization antibodies coexisted in pigs infected with the Japanese strains, presenting challenges for control measures.

Avian retroviral cardiomyopathy induced by infectious molecular clones of avian leukosis viruses (fowl glioma-inducing virus variants).

We previously described cardiomyocyte abnormality caused by Km_5666 strain, a variant of fowl glioma-inducing virus (FGV) prototype, which is an avian leukosis virus (ALV). However, the cardiac involvement appeared to be eradicated from the flock after a few years. An epidemiological survey from 2017 to 2020 was performed to elucidate the current prevalence of the cardiopathogenic strains in this flock. Four of the 71 bantams pathologically examined showed both glioma and cardiomyocyte abnormality, from which three ALV strains were detected. DNA sequencing revealed that several different ALV strains coexisted in each bantam and that the conserved Km_5666 virus fluid also contained at least two different ALV strains. We generated three infectious molecular clones from these samples, named KmN_77_clone_A, KmN_77_clone_B, and Km_5666_clone. The envSU of KmN_77_clone_A shared high sequence identity with that of Km_5666 (94.1%). In contrast, the envSU of KmN_77_clone_B showed >99.2% nucleotide similarity with that of an FGV variant without cardiopathogenicity. Furthermore, Km_5666_clone experimentally reproduced both gliomas and cardiomyocyte abnormality in chickens. From these results, it is suggested that the pathogenic determinant of cardiomyocyte abnormality is located in envSU similar to that of Km_5666. The cloning technique described here is beneficial for evaluating the viral pathogenicity in cases where affected birds are coinfected with several different ALV strains.

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.

A critical review on experimental Streptococcus suis infection in pigs with a focus on clinical monitoring and refinement strategies.

Streptococcus suis (S. suis) is a major pig pathogen worldwide with zoonotic potential. Though different research groups have contributed to a better understanding of the pathogenesis of S. suis infections in recent years, there are still numerous neglected research topics requiring animal infection trials. Of note, animal experiments are crucial to develop a cross-protective vaccine which is highly needed in the field. Due to the severe clinical signs associated with S. suis pathologies such as meningitis and arthritis, implementation of refinement is very important to reduce pain and distress of experimentally infected pigs. This review highlights the great diversity of clinical signs and courses of disease after experimental S. suis pig infections. We review clinical read out parameters and refinement strategies in experimental S. suis pig infections published between 2000 and 2021. Currently, substantial differences exist in describing clinical monitoring and humane endpoints. Most of the reviewed studies set the body temperature threshold of fever as high as 40.5°C. Monitoring intervals vary mainly between daily, twice a day and three times a day. Only a few studies apply scoring systems. Published scoring systems are inconsistent in their inclusion of parameters such as body temperature, feeding behavior, and respiratory signs. Locomotion and central nervous system signs are more common clinical scoring parameters in different studies by various research groups. As the heterogenicity in clinical monitoring limits the comparability between studies we hope to initiate a discussion with this review leading to an agreement on clinical read out parameters and monitoring intervals among S. suis research groups.

Experimental infection of Aedes (Stegomyia) albopictus and Culex pipiens mosquitoes with Bluetongue virus.

Bluetongue disease (BT), caused by Bluetongue virus (BTV), infects wild and domestic ruminants, causing severe economic damage in the cattle and sheep industry. Proven vectors of BTV are biting midges belonging to the Culicoides genus, but other arthropods are considered potential vectors, such as ticks, mosquitoes, wingless flies, and sand flies. The present study represents the first attempt to evaluate the vectorial capacity of Culex pipiens and Aedes albopictus for BTV. Mosquitoes were artificially fed with blood containing BTV serotype 1. Infection, dissemination and transmission rates were evaluated at 0, 3, 7, 14 and 21 days after an infected blood meal. Viral RNA was only detected up to 3 days post infection in the bodies of both species. This study indicates that the two Italian populations of Cx. pipiens and Ae. albopictus are not susceptible to BTV infection.

Experimental acute Clostridium perfringens type D enterotoxemia in sheep is not characterized by specific renal lesions.

Type D enterotoxemia, caused by Clostridium perfringens epsilon toxin (ETX), is one of the most economically important clostridial diseases of sheep. Acute type D enterotoxemia is characterized by well-documented lesions in the nervous, cardiocirculatory, and pulmonary systems. However, discrepancies and confusion exist as to whether renal lesions are part of the spectrum of lesions of this condition, which is controversial considering that for many decades it has been colloquially referred to as "pulpy kidney disease." Here, the authors assess renal changes in an experimental model of acute type D enterotoxemia in sheep and evaluate the possible role of ETX in their genesis. Four groups of 6 sheep each were intraduodenally inoculated with either a wild-type virulent C. perfringens type D strain, an etx knockout mutant unable to produce ETX, the etx mutant strain complemented with the wild-type etx gene that regains the ETX toxin production, or sterile culture medium (control group). All sheep were autopsied less than 24 hours after inoculation; none of them developed gross lesions in the kidneys. Ten predefined histologic renal changes were scored in each sheep. The proportion of sheep with microscopic changes and their severity scores did not differ significantly between groups. Mild intratubular medullary hemorrhage was observed in only 2 of the 12 sheep inoculated with the wild-type or etx-complemented bacterial strains, but not in the 12 sheep of the other 2 groups. The authors conclude that no specific gross or histologic renal lesions are observed in sheep with experimental acute type D enterotoxemia.

A laboratory challenge model for evaluating enyterocytozoon hepatopenaei susceptibility in selected lines of pacific whiteleg shrimp Penaeus vannamei.

Here we report for the first time a laboratory challenge model for Enterocytozoon hepatopenaei (EHP) to determine the difference of two Specific Pathogen Free (SPF) lines of Penaeus vannamei shrimp. These lines were experimentally challenged using EHP-infected fecal strings as inoculum. Real-time PCR and histopathology assays were performed to confirm EHP infection and evaluate differences in EHP susceptibility in the two genetic lines screened. Although the histopathology of the hepatopancreas tissue showed EHP lesions in both challenged groups, the histological lesions were more pronounced in one of the SPF lines. Quantitative PCR results revealed that animals displaying less hepatocellular damage have lower EHP load compared to animals displaying more pronounced pathological changes. There was no significant difference in final survival at 36 days post-infection in these lines with survival ranging between 80 and 100%. The data showed that mortality as an endpoint metric is not a suitable parameter to determine genetic susceptibility to EHP. Instead, histopathological changes in hepatopancreas, EHP load of the same tissue, and growth retardation would be better metrics to screen EHP susceptibility in P. vannamei. The results show the feasibility of screening genetic lines of P. vannamei for EHP resistance/tolerance using fecal string as an inoculum and, assessing histopathological changes, EHP load, and weight as indicators of resistance.

Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached nearly every country in the world with extraordinary person-to-person transmission. The most likely original source of the virus was spillover from an animal reservoir and subsequent adaptation to humans sometime during the winter of 2019 in Wuhan Province, China. Because of its genetic similarity to SARS-CoV-1, it is probable that this novel virus has a similar host range and receptor specificity. Due to concern for human-pet transmission, we investigated the susceptibility of domestic cats and dogs to infection and potential for infected cats to transmit to naive cats. We report that cats are highly susceptible to infection, with a prolonged period of oral and nasal viral shedding that is not accompanied by clinical signs, and are capable of direct contact transmission to other cats. These studies confirm that cats are susceptible to productive SARS-CoV-2 infection, but are unlikely to develop clinical disease. Further, we document that cats developed a robust neutralizing antibody response that prevented reinfection following a second viral challenge. Conversely, we found that dogs do not shed virus following infection but do seroconvert and mount an antiviral neutralizing antibody response. There is currently no evidence that cats or dogs play a significant role in human infection; however, reverse zoonosis is possible if infected owners expose their domestic pets to the virus during acute infection. Resistance to reinfection holds promise that a vaccine strategy may protect cats and, by extension, humans.

Toxocara tanuki larval distribution in mice and the infectivity of tissue larvae.

Toxocara tanuki is a common large roundworm in raccoon dogs. Experimental infection studies of T. tanuki in mice were conducted to clarify the distribution and infectivity of larvae in tissue. Groups of BALB/c and C57BL/6 mice (n = 5 mice/group) were each inoculated with 1000 embryonated T. tanuki eggs and necropsied at 7, 31, 91, and 182 days post inoculation (dpi). The number of larvae in the central nervous system, heart, lungs, kidneys, spleen, gastrointestinal tract, liver, and carcass was examined. Larvae obtained from the aforementioned mice on different days of the necropsy were orally inoculated into four groups of ICR mice (n = 6 mice/group) that were then necropsied at 21 dpi. Larvae were recovered from all mice. In the BALB/c and C57BL/6 mice, most of the larvae (> 88.7%) were recovered from the liver and the remainder from other tissues. The total number of larvae recovered from C57BL/6 mice was significantly higher than that from BALB/c mice, but no difference in the relative larval distribution within the viscera between the two mouse strains was observed. The mean recovery percentage of larvae from ICR mice infected with 182-day-old tissue larvae was 3.3%. Our findings showed that T. tanuki larvae migrated predominantly to the liver of mice and that the larvae maintained their infectivity for at least half a year.

Bacteriocin-Producing Escherichia coli Q5 and C41 with Potential Probiotic Properties: In Silico, In Vitro, and In Vivo Studies.

Commensal bacteriocin-producing Escherichia coli are of interest for possible use as probiotics to selectively control the spread of pathogenic bacteria. Here, we evaluated the biosafety and efficacy of two new bacteriocin-producing E. coli strains, Q5 (VKM B-3706D) and C41 (VKM B-3707D), isolated from healthy farm animals. The genomes of both strains were sequenced, and genes responsible for the antagonistic and colonization abilities of each strain were identified. In vitro studies have shown that both strains were medium-adhesive and demonstrated antagonistic activity against most enteropathogens tested. Oral administration of 5 × 108 to 5 × 1010 colony-forming units of both strains to rats with drinking water did not cause any disease symptoms or side effects. Short-term (5 days) oral administration of both strains protected rats from colonization and pathogenic effects of a toxigenic beta-lactam-resistant strain of E. coli C55 and helped preserve intestinal homeostasis. Taken together, these in silico, in vitro, and in vivo data indicate that both strains (and especially E. coli Q5) can be potentially used for the prevention of colibacillosis in farm animals.

Sheep and goat response to Trichostrongylus colubriformis infection based on egg output and worm burden.

This study was aimed to assess the performance of Trichostrongylus colubriformis and the host's responses to regulate egg outputs and worm burden. Infective larva (L3) was prepared by culturing egg of the worms harvested from the intestine of slaughtered sheep. Following this, L3 was maintained in the donor sheep to get adequate quantity of it for experimental trials. A complete randomized block design was used by considering host as blocking factor. Twenty eight small ruminants (sheep = 14) and (goat = 14) were employed in such a way that half of them were drenched with 10,000 T. colubriformis L3 and the remaining halves were controls. Faecal egg count (FEC) was recorded from the initial period (day-zero) up to day-56. At the end of experiment, animals were euthanized in humane manner, worms were recovered from the intestines, counted, and burden was estimated. FEC at various days post infection was insignificantly (P > 0.05) higher in goats than in sheep. The worm burden was significantly (P = 0.040) higher in infected goat than in infected sheep despite they were given equal dose of L3. In conclusion, relatively lower worm burden under natural conditions in goat may be due to their feeding behavior rather than their inherent resistance.

The Controlled Human Infection Model for Enterotoxigenic Escherichia coli.

The controlled human infection model (CHIM) for enterotoxigenic Escherichia coli (ETEC) has been instrumental in defining ETEC as a causative agent of acute watery diarrhea, providing insights into disease pathogenesis and resistance to illness, and enabling preliminary efficacy evaluations for numerous products including vaccines, immunoprophylactics, and drugs. Over a dozen strains have been evaluated to date, with a spectrum of clinical signs and symptoms that appear to replicate the clinical illness seen with naturally occurring ETEC. Recent advancements in the ETEC CHIM have enhanced the characterization of clinical, immunological, and microbiological outcomes. It is anticipated that omics-based technologies applied to ETEC CHIMs will continue to broaden our understanding of host-pathogen interactions and facilitate the development of primary and secondary prevention strategies.

Risk of transmission of foot-and-mouth disease by wild animals: infection dynamics in Japanese wild boar following direct inoculation or contact exposure.

Understanding of disease dynamics and viral shedding in wild boar and of the potential for disease spreading within wild boar and domestic pig populations is critical for developing effective control and eradication measures for foot-and-mouth disease (FMD). Accordingly, we infected experimentally wild boar and domestic pigs with FMD virus (FMDV) strains O/TAI/315/2016 and A/MOG/2013, and studied their susceptibility and viral transmissibility in both populations. Similar to FMDV-infected pigs, wild boar inoculated with both viruses exhibited vesicular lesions on their feet, snout, tongue and lip, although they did not show lameness. Further, inoculated wild boar were equally capable of transmitting the virus to all of their contact animals. While all contact pigs developed vesicular lesions after contact with inoculated animals, in contrast, no wild boar when exposed to the same infected animals showed obvious clinical signs. These results will be useful for further understanding of the critical roles in occurring and sustaining an FMD outbreak, and will be useful for establishing epidemiological surveillance programs and effective countermeasures for wild boar.