Highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b from Peru forms a monophyletic group with Chilean isolates in South America.

Highly pathogenic avian Influenza virus (HPAIV) has spread in an unprecedented extent globally in recent years. Despite the large reports of cases in Asia, Europe, and North America, little is known about its circulation in South America. Here, we describe the isolation, and whole genome characterization of HPAIV obtained from sampling 26 wild bird species in Peru, representing one of the largest studies in our region following the latest HPAIV introduction in South America. Out of 147 samples analyzed, 22 were positive for detection of avian influenza virus using a qRT-PCR-based assay. Following inoculation into embryonated chicken eggs, fourteen viral isolates were obtained from which nine isolates were selected for genome characterization, based on their host relevance. Our results identified the presence of HPAIV H5N1 subtype in a highly diverse wild bird species. Phylogenetic analysis revealed that these isolates correspond to the clade 2.3.4.4b, sharing a common ancestor with North American isolates and forming a monophyletic group along with isolates from Chile. Altogether, changes at the amino acid levels compared to their closest relatives indicates the virus is evolving locally, highlighting the need for constant genomic surveillance. This data evidence the chances for spillover events increases as the virus spreads into large populations of immunologically naïve avian species and adding conditions for cross species transmission.

Evaluation of the antiviral activity of ultraviolet light and zinc oxide nanoparticles on textile products exposed to Avian coronavirus.

This research has developed a piece of sanitizing locker-model equipment for textiles exposed to avian coronavirus, which has been put under the influence of UV light, UV + zinc oxide nanoparticles (phytosynthesized ZnONP), and water + UV, and, in turn, under the influence of the exposure time (60, 120, 180 s). The results linked to the phytosynthesis of ZnONP indicate a novel method of fabricating nanostructured material, nanoparticles with spherical morphology and an average size of 30 nm. The assays were made based on the viral viability of avian coronavirus according to the mortality of SPF embryonated eggs and a Real-Time PCR for viral load estimation. This was a model to evaluate the sanitizing effects against coronaviruses since they share a very similar structure and chemistry with SAR-CoV-2. The influence of the type of textile treatment evidenced the potential effect of the sanitizing UV light, which achieved 100% of embryo viability. The response of the ZnONP + UV nebulization showed a notorious influence of photoactivation according to the exposure time, and the 60-s treatment achieved a decrease of 88.9% in viral viability, compared to 77.8% and 55.6% corresponding to the 120 and 180-s treatments, respectively. Regarding the decrease in viral load between the types of treatments, UV 180 s reduced 98.42% and UV 60 s + ZnONP reduced 99.46%, respectively. The results show the combinatorial effect of UV light and zinc nanoparticles in decreasing the viral viability of avian coronavirus, as a model of other important coronaviruses in public health such as SARS-CoV-2.

Genetic Analysis of Infectious Bronchitis Virus S1 Gene Reveals Novel Amino Acid Changes in the GI-16 Lineage in Peru.

Infectious bronchitis is a highly contagious viral disease that represents an economic threat for poultry despite the wide use of vaccination. To characterize the virus circulating in Peru, we analyzed 200 samples, including nasopharyngeal swabs and multiple tissues collected from animals suspected of being infected with infectious bronchitis virus (IBV) between January and August in 2015. All animals had at least one positive sample for IBV by RT-PCR. Out of these positive samples, eighteen (18) were selected for viral isolation and a partial S1 sequencing. Phylogenetic analysis showed that sixteen isolates clustered with members of GI-16 lineage, also known as Q1, with nucleotide homology ranging from 93% to 98%. The two remaining isolates grouped with members of the GI-1 lineage. Our study reveals circulation of GI-16 lineage during this period in poultry systems in Peru, along with GI-1 lineage (vaccine-derived). Moreover, those IBV GI-16 isolates showed unique nucleotide and amino acid changes compared to their closest relatives. Altogether, these findings reveal the circulation of GI-16 lineage while describing changes at key regions of the S protein that might be of relevance for vaccine evasion. These results highlight the importance of genetic surveillance for improving vaccination strategies against infectious bronchitis.

Genetic subtyping and phylogenetic analysis of HA and NA from avian influenza virus in wild birds from Peru reveals unique features among circulating strains in America.

Avian influenza virus (AIV) represents a major concern with productive implications in poultry systems but it is also a zoonotic agent that possesses an intrinsic pandemic risk. AIV is an enveloped, negative-sense and single-stranded RNA virus with a segmented genome. The eight genomic segments, comprising the whole genome, encode for eleven proteins. Within these proteins, Hemagglutinin (HA) and Neuraminidase (NA) are the most relevant for studies of evolution and pathogenesis considering their role in viral replication, and have also been used for classification purposes. Migratory birds are the main hosts and play a pivotal role in viral evolution and dissemination due to their migratory routes that comprise large regions worldwide. Altogether, viral and reservoir factors contribute to the emergence of avian influenza viruses with novel features and pathogenic potentials. The study aimed to conduct surveillance of AIVs in wild birds from Peru. A multi-site screening of feces of migratory birds was performed to isolate viruses and to characterize the whole genome sequences, especially the genes coding for HA and NA proteins. Four-hundred-twenty-one (421) fecal samples, collected between March 2019 and March 2020 in Lima, were obtained from 21 species of wild birds. From these, we isolated five AIV from whimbrel, kelp gull, Franklin's gulls and Mallard, which were of low pathogenicity, including four subtypes as H6N8, H13N6, H6N2 and H2N6. Genetic analysis of HA and NA genes revealed novel features in these viruses and phylogenetic analysis exhibited a close relationship with those identified in North America (US and Canada). Furthermore, H2N6 isolate presented a NA sequence with higher genetic relationship to Chilean isolates. These results highlight that the geographical factor is of major relevance in the evolution of AIV, suggesting that AIV circulating in Peru might represent a new site for the emergence of reassortant AIVs.

First isolation and whole genome characterization of porcine deltacoronavirus from pigs in Peru.

Porcine deltacoronavirus is a newly emergent enteric pathogen affecting swine farms worldwide. It has been detected in several countries in Europe, Asia and North America; yet, it has not been reported in South America. In November 2019, an enteric disease outbreak in a pig farm located in San Martin, Peru, was reported along with submission of three intestinal samples from pigs who succumbed to the disease. Samples were processed for molecular detection by qRT-PCR, viral isolation and further sequencing analysis. A taqman-based RT-PCR was performed to differentiate among the most relevant swine enteric coronaviruses described to date. All samples were positive to porcine deltacoronavirus with a cycle threshold (Ct) value between 9 and 14, revealing a high viral load, while testing negative to porcine epidemic diarrhea and transmissible gastroenteritis viruses. Following detection, viral isolation was performed using PK-15 and Vero cell lines. After 5 days of inoculation, no cytopathic effect was observed. A second blind passage allowed the observation of cytopathic effect on PK-15 cells, while it remained absent in Vero cells. A fluorescence test using an anti-N monoclonal antibody confirmed viral replication. One sample was processed for whole genome sequencing (WGS). In short, raw reads were imported into CLC genomics and assembled de novo. Out of 479k reads generated from the sample, 436k assembled into a 25,501 bp contig which was 99.5% identical to a reference porcine deltacoronavirus strain from the USA within the North American phylogroup. Yet, there are relevant differences at the nucleotide and amino acid levels compared with previously described porcine deltacoronavirus strains. Altogether, our findings represent the first report of porcine deltacoronavirus in South America, which provides information of its evolutionary origin. Thus, this study offers new insights into the molecular epidemiology of porcine deltacoronavirus infections in the swine industry.

Differential Effects of Drinking Water Quality on Phagocyte Responses of Broiler Chickens Against Fungal and Bacterial Challenges.

Combinatorial effects of xenobiotics in water on health may occur even at levels within current acceptable guidelines for individual chemicals. Herein, we took advantage of the sensitivity of the immune system and an avian animal model to examine the impact of xenobiotic mixtures on animal health. Water was derived from an underground well in Alberta, Canada and met guidelines for consumption, but contained a number of contaminants. Changes to chicken immunity were evaluated following acute (7d) exposure to contaminated water under basal and immune challenged conditions. An increase in resident macrophages and a decrease in CD8+ lymphocytes were identified in the abdominal cavity, which served as a relevant site where immune leukocytes could be examined. Subsequent intra-abdominal immune stimulation detected differential in vivo acute inflammatory responses to fungal and bacterial challenges. Leukocyte recruitment into the challenge site and activation of phagocyte antimicrobial responses were affected. These functional responses paralleled molecular changes in the expression for pro-inflammatory and regulatory genes. In all, this study primarily highlights dysregulation of phagocyte responses following acute (7d) exposure of poultry to contaminated water. Given that production food animals hold a unique position at the interface of animal, environmental and human health, this emphasizes the need to consider the impact of xenobiotic mixtures in our assessments of water quality.

Neonatal Exposure to Amoxicillin Alters Long-Term Immune Response Despite Transient Effects on Gut-Microbiota in Piglets.

Antibiotic exposure during neonatal development may result in transient or persistent alterations of key microbes that are vital for normal development of local and systemic immunity, potentially impairing immune competence later in life. To further elucidate the relationship between antibiotic exposure and immune development, newborn pigs were exposed to a therapeutic pediatric dose (30 mg/kg/day) of amoxicillin (AB) or placebo (PL) from post-natal day (PND) 0-14. Subsequently, immune cell phenotype, microbial composition, and immune response to an intraperitoneal (IP) challenge with Salmonella enterica serovar Typhimurium were evaluated. AB exposure caused significant changes in fecal microbial composition on PND 3 (P = 0.025). This stemmed from a 2-fold increase in Enterobacteriaceae with live cecal coliforms on PND 7 indicating at 10-fold increase (P = 0.036). Alterations in microbial composition were transient, and successional patterns were normalizing by PND 14 (P = 0.693). Differences in PBMC (peripheral blood mononuclear cell) immune cell subtypes were detected, with the percentage of CD3+CD4+ T cells among the broader T cell population (CD3+CD4+/CD3+) being significantly higher (P = 0.031) in AB pigs and the numbers of CD4+CD45RA+ (naïve) T cells per liter of blood were lower on PND 21 in AB pigs (P = 0.036). Meanwhile, PBMCs from AB pigs produced significantly more IFNγ upon stimulation with a T-cell mitogen on PND 21 and 49 (P = 0.021). When AB pigs were challenged with heat-killed Salmonella (IP) on PND 49, IFNγ gene expression in peripheral blood was upregulated compared to those treated with PL (P = 0.043). Additionally, AB pigs showed stronger activation among neutrophils infiltrating the peritoneal cavity after in vivo immune challenge, based on higher levels of NF-κB nuclear translocation (P = 0.001). Overall, our results indicate that early life treatment with a therapeutically relevant dose of a commonly prescribed antibiotic has a programming effect on the immune system. Despite antibiotics only causing a transient disruption in gut-associated microbial communities, implications were long-term, with antibiotic treated pigs mounting an upregulated response to an immune challenge. This research adds to the growing body of evidence indicating adverse immune outcomes of early life antibiotic exposures.

Identification of the First Teleost CD5 Molecule: Additional Evidence on Phenotypical and Functional Similarities between Fish IgM+ B Cells and Mammalian B1 Cells.

Despite teleost fish being the first animal group in which all elements of adaptive immunity are present, the lack of follicular structures, as well as the fact that systemic Ab responses rely exclusively on unswitched low-affinity IgM responses, strongly suggests that fish B cell responses resemble mammalian B1 cell responses rather than those of B2 cells. In line with this hypothesis, in the current study, we have identified a homolog of CD5 in teleost fish. This pan-T marker belonging to the scavenger receptor cysteine-rich family of receptors is commonly used in mammals to distinguish a subset of B1 cells. Subsequently, we have demonstrated that a very high percentage of teleost IgM+ B cells express this marker, in contrast to the limited population of CD5-expressing B1 cells found in most mammals. Furthermore, we demonstrate that fish IgM+ B cells share classical phenotypic features of mammalian B1 cells such as large size, high complexity, high surface IgM, and low surface IgD expression, regardless of CD5 expression. Additionally, fish IgM+ B cells, unlike murine B2 cells, also displayed extended survival in cell culture and did not proliferate after BCR engagement. Altogether, our results demonstrate that although fish are evolutionarily the first group in which all the elements of acquired immunity are present, in the absence of follicular structures, most teleost IgM+ B cells have retained phenotypical and functional characteristics of mammalian B1 cells.

Innate Immunity Provides Biomarkers of Health for Teleosts Exposed to Nanoparticles.

In recent years, the unique properties of nanoparticles have fostered novel applications in various fields such as biology, pharmaceuticals, agriculture, and others. Unfortunately, their rapid integration into daily life has also led to environmental concerns due to uncontrolled release of nanoparticles into the aquatic environment. Despite increasing awareness of nanoparticle bioaccumulation in the aquatic environment, much remains to be learned about their impact on aquatic organisms and how to best monitor these effects. Herein, we provide the first review of innate immunity as an emerging tool to assess the health of fish following nanoparticle exposure. Fish are widely used as sentinels for aquatic ecosystem pollution and innate immune parameters offer sensitive and reliable tools that can be harnessed for evaluation of contamination events. The most frequent biomarkers highlighted in literature to date include, but are not limited to, parameters associated with leukocyte dynamics, oxidative stress, and cytokine production. Taken together, innate immunity offers finite and sensitive biomarkers for assessment of the impact of nanoparticles on fish health.

Contribution of leukocytes to the induction and resolution of the acute inflammatory response in chickens.

A successful immune response against invading pathogens relies on the efficient activation of host defense mechanisms and a timely return to immune homeostasis. Despite their importance, these mechanisms remain ill-defined in most animal groups. This study focuses on the acute inflammatory response of chickens, important both as an avian model with a unique position in evolution as well as an increasingly notable target of infectious zoonotic diseases. We took advantage of an in vivo self-resolving intra-abdominal challenge model to provide an integrative view of leukocyte responses during the induction and resolution phases of acute inflammation. Our results showed rapid leukocyte infiltration into the abdominal cavity post zymosan challenge (significant increase as early as 4 h), which was dominated by heterophils. Peak leukocyte infiltration and ROS production reached maximum levels at 12 h post challenge, which was significantly earlier than comparative studies in teleost fish and mice. Both heterophils and monocyte/macrophages contributed to ROS production. Local leukocyte infiltration was preceded by an increase in peripheral leukocytes and a drop in the number of bone marrow leukocytes. The proportion of apoptotic leukocytes increased following peak of acute inflammation, rising to significant levels within the abdominal cavity by 48 h, consistent with other indicators for the resolution of inflammation. Importantly, comparison of chicken phagocytic responses with those previously shown in agnathan, teleost and murine models suggested a progressive evolutionary shift towards an increased sensitivity to pro-inflammatory pathogen-derived particles and decreased sensitivity towards homeostatic stimuli. Thus, while significant conservation can be noted across the immune systems of endotherms, this study highlights additional unique features that govern the induction and resolution of acute inflammation in the avian system, which may be relevant to disease susceptibility and performance.

Application of imaging flow cytometry for characterization of acute inflammation in non-classical animal model systems.

Phagocytes display marked heterogeneity in their capacity to induce and control acute inflammation. This has a significant impact on the effectiveness of antimicrobial immune responses at different tissue sites as well as their predisposition for inflammation-associated pathology. Imaging flow cytometry provides novel opportunities for characterization of these phagocyte populations through high spatial resolution, statistical robustness, and a broad range of quantitative morphometric cell analysis tools. This study highlights an integrative approach that brings together new tools in imaging flow cytometry with conventional methodologies for characterization of phagocyte responses during acute inflammation. We focus on a comparative avian in vivo challenge model to showcase the added depth gained through these novel quantitative multiparametric approaches even in the absence of antibody-based cellular markers. Our characterization of acute inflammation in this model shows significant conservation of phagocytic capacity among avian phagocytes compared to other animal models. However, it also highlights evolutionary divergence with regards to phagocyte inflammation control mechanisms based on the internalization of apoptotic cells.

Multi-parametric analysis of phagocyte antimicrobial responses using imaging flow cytometry.

We feature a multi-parametric approach based on an imaging flow cytometry platform for examining phagocyte antimicrobial responses against the gram-negative bacterium Aeromonas veronii. This pathogen is known to induce strong inflammatory responses across a broad range of animal species, including humans. We examined the contribution of A. veronii to the induction of early phagocyte inflammatory processes in RAW 264.7 murine macrophages in vitro. We found that A. veronii, both in live or heat-killed forms, induced similar levels of macrophage activation based on NF-κB translocation. Although these macrophages maintained high levels of viability following heat-killed or live challenges with A. veronii, we identified inhibition of macrophage proliferation as early as 1h post in vitro challenge. The characterization of phagocytic responses showed a time-dependent increase in phagocytosis upon A. veronii challenge, which was paired with a robust induction of intracellular respiratory burst responses. Interestingly, despite the overall increase in the production of reactive oxygen species (ROS) among RAW 264.7 macrophages, we found a significant reduction in the production of ROS among the macrophage subset that had bound A. veronii. Phagocytic uptake of the pathogen further decreased ROS production levels, even beyond those of unstimulated controls. Overall, this multi-parametric imaging flow cytometry-based approach allowed for segregation of unique phagocyte sub-populations and examination of their downstream antimicrobial responses, and should contribute to improved understanding of phagocyte responses against Aeromonas and other pathogens.

Seroprevalencia del virus de la diarrea viral en bovinos de crianza extensiva de la provincia de San Pablo, Cajamarca / Seroprevalence of bovine viral diarrhea virus in grazing cattle in Cajamarca, Peru

El objetivo del presente estudio fue determinar la seroprevalencia del virus de la Diarrea Viral Bovina (VDVB) en bovinos criollos de crianza extensiva, sin historia de vacunación, en la provincia de San Pablo, Cajamarca. Se emplearon 385 muestras del banco de sueros, colectadas en el 2004. Las muestras se estratificaron en cuatro grupos etarios (2 a 24 meses) y por sexo. La detección de anticuerpos contra el VDVB se hizo mediante la prueba de neutralización viral. El 27.1 ± 4.4% (104/385) de los bovinos presentó anticuerpos contra el VDVB indistintamente del grupo etario o sexo; sin embargo, el 71.2 ± 8.7% (47/66) de los animales entre 12 y 24 meses de edad presentaron títulos de anticuerpos entre 128 a >256. Se concluye que el VDVB está presente con una prevalencia baja en la población de bovinos de la provincia de SanPablo.

The aim of this study was to determine the seroprevalence of Bovine Viral Diarrhea virus (BVDV) in grazing cattle without history of vaccination, in the province of San Pablo, Cajamarca, Peru. It was used 385 samples from the serum bank, collected in 2004. Samples were stratified in four age groups (2 to 24 months) and by sex. The detection of antibodies against BVDV was done by the viral neutralization test. The 27.0 ± 4.4% (104/385) of samples had antibodies against BVDV, and without statistical difference due to age or sex; however, 71.2 ± 8.7% (47/66) of animals between 12 and 24 months of age showed antibody titres between 128 and >256. It was concluded that the BVDV is present with a low seroprevalence in the cattle population of San Pablo province, Cajamarca.