Emerging Pandemic Diseases: How We Got to COVID-19.

Infectious diseases prevalent in humans and animals are caused by pathogens that once emerged from other animal hosts. In addition to these established infections, new infectious diseases periodically emerge. In extreme cases they may cause pandemics such as COVID-19; in other cases, dead-end infections or smaller epidemics result. Established diseases may also re-emerge, for example by extending geographically or by becoming more transmissible or more pathogenic. Disease emergence reflects dynamic balances and imbalances, within complex globally distributed ecosystems comprising humans, animals, pathogens, and the environment. Understanding these variables is a necessary step in controlling future devastating disease emergences.

Characterization of Cetacean Morbillivirus in Humpback Whales, Brazil.

Cetacean morbillivirus is an etiologic agent associated with strandings of live and dead cetacean species occurring sporadically or as epizootics worldwide. We report 2 cases of cetacean morbillivirus in humpback whales (Megaptera novaeangliae) in Brazil and describe the anatomopathological, immunohistochemical, and molecular characterization findings in the specimens.

Development of a Multiplex Real-Time PCR to Disambiguate Culicoides sonorensis within Culicoides variipennis Complex, the Proven Vector of Bluetongue and Epizootic Hemorrhagic Disease Viruses in North America.

Species delimitation of Culicoides complex species can be challenging. Among species within the Culicoides variipennis complex, C. sonorensis is considered the primary vector of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in North America. Morphological identification of C. sonorensis within the C. variipennis complex is laborious, time-consuming, and requires entomology expertise. Therefore, in this study we developed and validated a multiplex real-time PCR for rapid detection and differentiation of C. sonorensis from the two other main cryptic species (C. variipennis and C. occidentalis) within the C. variipennis complex. The assay targets the EF1α gene and has a built-in internal control targeting 18 S. The specificity and the sensitivity of the multiplex real-time PCR were evaluated using morphologically identified reference and field-collected specimens. The multiplex PCR was 100% specific when nucleic acid extracted from C. variipennis, sonorensis, and occidentalis specimens was tested. When nucleic acid extracted from pools of midges was tested, the multiplex PCR was able to detect all three Culicoides species with comparable sensitivity. The multiplex assay, however, failed to detect eight morphologically identified C. sonorensis specimens collected from Alberta in 2014. The EF1α gene sequences of these specimens formed a distinct phylogenetic cluster, amongst those from C. variipennis, sonorensis, and occidentalis, suggesting that they belong to a different species. We hypothesize that those specimens might be C. albertensis, the only other species remaining in the C. variipennis complex with known geographical distribution in North America. We believe that this highly sensitive and specific multiplex real-time PCR assay could be an effective tool for rapid detection and differentiation of C. sonorensis, the known vector of BTV and EHDV, in trap collections in future vector surveillance programs.

Molecular characterisation of Histoplasma capsulatum sensu lato from Ethiopian horses reveals two distinct phylogenetic clades.

Epizootic lymphangitis (EL) is a highly prevalent and contagious infectious disease affecting horses in many parts of Ethiopia caused by Histoplasma capsulatum sensu lato ('var. farciminosum'). In this study, 12 suspected isolates of H. capsulatum sensu lato or yeasts unidentified by conventional biochemical tests isolated from Ethiopian horses with EL were characterised by internal transcribed spacer sequencing. Six of the 12 isolates were identified to be members of H. capsulatum sensu lato and the other six were Pichia kudriavzevii (synonym: Candida krusei) (n = 3), Trichosporon asahii (n = 1), Geotrichum silvicola (n = 1) and Moesziomyces aphidis (n = 1), respectively. The six H. capsulatum sensu lato isolates were further characterised by multilocus sequence analysis. Four distinct gene loci (arf [462 bases], H-anti [410 bases], ole1 [338 bases] and tub1 [272 bases]) of these six isolates as well as those of two H. capsulatum sensu lato ('var. farciminosum') reference strains (ATCC 58332 and ATCC 28798) were polymerase chain reaction (PCR)-amplified and sequenced. Phylogenetic analyses of their concatenated nucleotide sequences showed that three of the isolates and the reference strain ATCC 58332 were identical and belonged to the Eurasia clade within Latin American (LAm) A (H. suramericanum), and those of the other three isolates and the reference strain ATCC 28798 were identical and belonged to the Africa clade. At least two distinct phylogenetic clades of H. capsulatum sensu lato were circulating in Ethiopian horses with EL. Advanced molecular technologies and bioinformatics tools are crucial for the accurate identification and typing of pathogens as well as the discovery of novel microorganisms in veterinary microbiology.

Using multilocus sequence analysis with four concatenated housekeeping gene loci, at least two distinct phylogenetic clades, namely Eurasia clade and Africa clade, of Histoplasma capsulatum sensu lato were confirmed to be circulating in Ethiopian horses with epizootic lymphangitis.

Growth inhibitory effect of selected medicinal plants from Southern Ethiopia on the mycelial phase of Histoplasma capsulatum var. farciminosum.

BACKGROUND: Epizootic lymphangitis is an infectious and chronically debilitating disease of the equines. Histoplasma capsulatum var. farciminosum, a thermally dimorphic fungi, is the causative agent for the disease. In Ethiopia, the disease significantly affects carthorses, posing threats to animal welfare, and resulting in substantial economic losses. Limited availability of widely accessible antifungals in addition to the chronic nature of the disease is the major challenge against management of epizootic lymphangitis. This study aimed to assess the in vitro efficacy of specific local medicinal plant extracts against the mycelial phase development of H. capsulatum var. farciminosum in southern Ethiopia. The leaves of Xanthium strumarium, Kanda (Family Rubiaceae), Croton macrostachyus (Bisana in Amharic), and Centella Asiatica (Echere waye as a local name in Zeyissegna) that are traditionally used for the treatment of different skin ailments were collected and extracted for the in vitro trial. RESULTS: The study revealed that methanol extracts of Xanthium strumarium, Kanda, Croton macrostachyus, and Centella Asiatica, at minimum inhibitory concentrations of 1.25 mg/ml, 2.5 mg/ml, 2.5 mg/ml, and 5 mg/ml, respectively, inhibited the growth of H. capsulatum var. farciminosum. CONCLUSION: This in vitro finding could serve as significant preliminary data in the exploration of effective alternative treatment options for epizootic lymphangitis. This study provides a crucial foundation for further research aimed at determining the chemical components and in vivo effectiveness of these plant extracts against both the mycelial and yeast forms of Histoplasma capsulatum var. farciminosum.

Outbreak of epizootic hemorrhagic disease in captive reindeer (Rangifer tarandus).

In September 2020, an outbreak of epizootic hemorrhagic disease occurred in captive reindeer (Rangifer tarandus) and was associated with neurological signs and mortality. Four reindeer died or were euthanized after acute illness over a 12-day period. Affected reindeer displayed abnormal behavior, neurologic signs, lethargy, and/or lameness. The most consistent gross finding was dark red streaks throughout the adrenal gland cortices (4/4). One animal had acute hemorrhage involving the subcutis and skeletal muscles over the ventrolateral body wall and back, and abomasal serosa. Histologically, the most common lesions were adrenal gland cortical hemorrhage (4/4) with necrosis (3/4) and lymphoplasmacytic meningoencephalitis with gliosis, glial nodules, satellitosis, and nonsuppurative perivascular cuffing (4/4). The brain lesions were most frequent in the gray matter of the cerebrum, hippocampus, and thalamus but also involved the cerebellum and brainstem. Epizootic hemorrhagic disease virus serotype 6 was detected through PCR and sequencing of the spleen in all cases.

Epizootic Hemorrhagic Disease Virus Serotype 8, Italy, 2022.

We describe the detection of epizootic hemorrhagic disease virus (EHDV) serotype 8 in cattle farms in Sardinia and Sicily in October-November 2022. The virus has a direct origin in North Africa; its genome is identical (>99.9% nucleotide sequence identity) to EHDV serotype 8 strains detected in Tunisia in 2021.

Host immune responses to enzootic and invasive pathogen lineages vary in magnitude, timing, and efficacy.

Infectious diseases of wildlife continue to pose a threat to biodiversity worldwide, yet pathogens are far from uniform in virulence or host disease outcome. Within the same pathogen species, virulence can vary considerably depending on strain or lineage, in turn eliciting variable host responses. One pathogen that has caused extensive biodiversity loss is the amphibian-killing fungus, Batrachochytrium dendrobatidis (Bd), which is comprised of a globally widespread hypervirulent lineage (Bd-GPL), and multiple geographically restricted, enzootic lineages. Whereas host immunogenomic responses to Bd-GPL have been characterized in a number of amphibian species, immunogenomic responses to geographically restricted, enzootic Bd lineages are less clear. To examine lineage-specific host immune responses to Bd, we exposed a species of pumpkin toadlet, Brachycephalus pitanga, which is endemic to Brazil's Southern Atlantic Forest, to either the Bd-GPL or the enzootic Bd-Asia-2/Brazil (hereafter Bd-Brazil) lineage. Using temporal samples from early, mid, and late infection stages, we quantified functional immunogenomic responses over the course of infection using differential gene expression tests and coexpression network analyses. Host immune responses varied significantly with Bd lineage. Relative to controls, toadlet responses to Bd-Brazil were weak at early infection (25 genes significantly differentially expressed), peaked by mid-stage infection (414 genes), and were nearly fully resolved by late-stage infection (nine genes). In contrast, responses to Bd-GPL were magnified and delayed; toadlets significantly differentially expressed 111 genes early, 87 genes at mid-stage infection, and 726 genes by late-stage infection relative to controls. Given that infection intensity did not vary between mid- and late-stage disease in either Bd-Brazil or Bd-GPL treatments, this suggests that pumpkin toadlets may be at least partially tolerant to the enzootic Bd-Brazil lineage. In contrast, late-stage immune activation against Bd-GPL was consistent with immune dysregulation previously observed in other species. Our results demonstrate that both the timing of immune response and the particular immune pathways activated are specific to Bd lineage. Within regions where multiple Bd lineages co-occur, and given continued global Bd movement, these differential host responses may influence not only individual disease outcome, but transmission dynamics at the population and community levels.

Modeling the role of land conversion on the spread of an epizootic disease.

Land conversion and the resulting contact between domesticated and wild species has arguably been the single largest contributor to the emergence of novel epizootic and zoonotic diseases in the past century. An unintended consequence of these interactions is zoonotic or epizootic disease spillovers from wild species to humans and their domesticates. Disease spillovers are edge effects of land conversion and are sensitive to the size and shape of converted areas. We combine spatial metrics from landscape ecology with theoretical epidemiological models to understand how the size and shape of land conversion affect epizootic and zoonotic disease transmission of single and two species populations. We show that the less compact the converted area, and the greater the depth of the contact zone, the more rapidly will an introduced disease spread through the domesticated population.

Mass mortality of collector urchins Tripneustes gratilla in Hawai`i.

As grazers, sea urchins are keystone species in tropical marine ecosystems, and their loss can have important ecological ramifications. Die-offs of urchins are frequently described, but their causes are often unclear, in part because systematic examinations of animal tissues at gross and microscopic level are not done. In some areas, urchins are being employed to control invasive marine algae. Here, we describe the pathology of a mortality event in Tripneustes gratilla in Hawai`i where urchins were translocated to control invasive algae. Although we did not determine the cause of the mortality event, our investigation indicates that animals died from inflammation of the test and epidermal ulceration, followed by inability to maintain coelomic fluid volume, colonization of coelomic fluid by opportunists (diatom, algae), and inappetence. Parasites, bacteria, fungi, and viruses were not evident as a primary cause of death. Pathology was suggestive of a toxin or other environmental cause such as lack of food, possibilities that could be pursued in future investigations. These findings highlight the need for caution and additional tools to better assess health when translocating marine invertebrates to ensure maximal biosecurity.

Temperature-associated virulence, species susceptibility and interspecies transmission of a Lactococcus petauri strain from rainbow trout.

Lactococcus petauri is an important emergent aquaculture pathogen in the USA. To better understand environmental conditions conducive to piscine lactococcosis and the susceptibility of fish species, laboratory-controlled challenges were used as models of infection. Rainbow trout Oncorhynchus mykiss maintained at 13 or 18°C were challenged by intracoelomic (ICe) injection with 101, 103 or 105 colony-forming units per fish (CFU fish-1) and monitored for 21 d. At 13°C, trout experienced mortalities of 7, 7 and 0%, and bacterial persistence of 0, 20 and 0% in survivors, respectively. When exposed to the same bacterial doses, trout maintained at 18°C experienced mortalities of 59, 84 and 91%, and bacterial persistence of 60, 66 and 0% in survivors, confirming a significant role of temperature in the pathogenesis of lactococcosis. Additionally, the susceptibility of rainbow trout, Chinook salmon Oncorhynchus tshawytscha, white sturgeon Acipenser transmontanus, Nile tilapia Oreochromis niloticus, and koi Cyprinus carpio to infection by L. petauri was compared using ICe challenges at 18°C. Trout and salmon experienced 96 and 56% cumulative mortality, respectively, and 17% of surviving salmon remained persistently infected. There were no mortalities in the other fish species, and no culturable bacteria recovered at the end of the challenge. However, when surviving fish were used in further cohabitation trials, naïve trout housed with previously exposed tilapia exhibited 6% mortality, demonstrating that non-salmonids can become sub-clinical carriers of this pathogen. The data obtained provide useful information regarding temperature-associated virulence, fish species susceptibility, and potential carrier transmission of L. petauri that can be used in the development of better management practices to protect against piscine lactococcosis.

Rapid onsets of warming events trigger mass mortality of coral reef fish.

Our study reveals a hitherto overlooked ecological threat of climate change. Studies of warming events in the ocean have typically focused on the events' maximum temperature and duration as the cause of devastating disturbances in coral reefs, kelp forests, and rocky shores. In this study, however, we found that the rate of onset (Ronset), rather than the peak, was the likely trigger of mass mortality of coral reef fishes in the Red Sea. Following a steep rise in water temperature (4.2 °C in 2.5 d), thermally stressed fish belonging to dozens of species became fatally infected by Streptococcus iniae Piscivores and benthivores were disproportionately impacted whereas zooplanktivores were spared. Mortality rates peaked 2 wk later, coinciding with a second warming event with extreme Ronset The epizootic lasted ∼2 mo, extending beyond the warming events through the consumption of pathogen-laden carcasses by uninfected fish. The warming was widespread, with an evident decline in wind speed, barometric pressure, and latent heat flux. A reassessment of past reports suggests that steep Ronset was also the probable trigger of mass mortalities of wild fish elsewhere. If the ongoing increase in the frequency and intensity of marine heat waves is associated with a corresponding increase in the frequency of extreme Ronset, calamities inflicted on coral reefs by the warming oceans may extend far beyond coral bleaching.

Emergence of epizootic hemorrhagic disease in Europe.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-borne disease transmitted by blood-sucking midges of the genus Culicoides. It affects domestic and wild ruminants, mainly white-tailed deer and cattle. At the end of October and in November 2022, outbreaks of EHD were confirmed in several cattle farms in Sardinia and Sicily. This is the first detection of EHD in Europe. The loss of free status and the lack of effective prophylactic measures could have significant economic consequences for infected countries.

[Emergence of epizootic hemorrhagic disease in Europe]. / Émergence de la maladie hémorragique épizootique en Europe.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-borne disease transmitted by blood-sucking midges of the genus Culicoides. It affects domestic and wild ruminants, mainly white-tailed deer and cattle. At the end of October and in November 2022, outbreaks of EHD were confirmed in several cattle farms in Sardinia and Sicily. This is the first detection of EHD in Europe. The loss of free status and the lack of effective prophylactic measures could have significant economic consequences for infected countries.

Mapping the Risk for West Nile Virus Transmission, Africa.

West Nile virus (WNV) is an emergent arthropodborne virus that is transmitted from bird to bird by mosquitoes. Spillover events occur when infected mosquitoes bite mammals. We created a geopositioned database of WNV presence in Africa and considered reports of the virus in all animal components: reservoirs, vectors, and nonhuman dead-end hosts. We built various biogeographic models to determine which drivers explain the distribution of WNV throughout Africa. Wetlands of international importance for birds accounted for the detection of WNV in all animal components, whereas human-related drivers played a key role in the epizootic cases. We combined these models to obtain an integrative and large-scale perspective of the areas at risk for WNV spillover. Understanding which areas pose the highest risk would enable us to address the management of this spreading disease and to comprehend the translocation of WNV outside Africa through avian migration routes.

Host-driven subspeciation in the hedgehog fungus, Trichophyton erinacei, an emerging cause of human dermatophytosis.

Trichophyton erinacei is a main cause of dermatophytosis in hedgehogs and is increasingly reported from human infections worldwide. This pathogen was originally described in the European hedgehog (Erinaceus europaeus) but is also frequently found in the African four-toed hedgehog (Atelerix albiventris), a popular pet animal worldwide. Little is known about the taxonomy and population genetics of this pathogen despite its increasing importance in clinical practice. Notably, whether there are different populations or even cryptic species associated with different hosts or geographic regions is not known. To answer these questions, we collected 161 isolates, performed phylogenetic and population-genetic analyses, determined mating-type, and characterised morphology and physiology. Multigene phylogeny and microsatellite analysis supported T. erinacei as a monophyletic species, in contrast to highly incongruent single-gene phylogenies. Two main subpopulations, one specific mainly to Atelerix and second to Erinaceus hosts, were identified inside T. erinacei, and slight differences in the size of microconidia and antifungal susceptibilities were observed among them. Although the process of speciation into two lineages is ongoing in T. erinacei, there is still gene flow between these populations. Thus, we present T. erinacei as a single species, with notable intraspecies variability in genotype and phenotype. The data from wild hedgehogs indicated that sexual reproduction in T. erinacei and de novo infection of hedgehogs from soil are probably rare events and that clonal horizontal spread strongly dominates. The molecular typing approach used in this study represents a suitable tool for further epidemiological surveillance of this emerging pathogen in both animals and humans. The results of this study also highlighted the need to use a multigene phylogeny ideally in combination with other independent molecular markers to understand the species boundaries of dermatophytes. Citation: Cmoková A, Kolarík M, Guillot J, et al. 2022. Host-driven subspeciation in the hedgehog fungus, Trichophyton erinacei, an emerging cause of human dermatophytosis. Persoonia 48: 203-218. https://doi.org/10.3767/persoonia.2022.48.06.

Highly Pathogenic Avian Influenza A(H5N8) Virus Spread by Short- and Long-Range Transmission, France, 2016-17.

We detected 3 genotypes of highly pathogenic avian influenza A(H5N8) virus in France during winter 2016-17. Genotype A viruses caused dramatic economic losses in the domestic duck farm industry in southwestern France. Our phylogenetic analysis suggests that genotype A viruses formed 5 distinct geographic clusters in southwestern France. In some clusters, local secondary transmission might have been started by a single introduction. The intensity of the viral spread seems to correspond to the density of duck holdings in each production area. To avoid the introduction of disease into an unaffected area, it is crucial that authorities limit the movements of potentially infected birds.

Constitutive low expression of antiviral effectors sensitizes melanoma cells to a novel oncolytic virus.

STAT1 is a critical effector and a target gene of interferon (IFN) signaling, and thus a central mediator of antiviral responses. As both a mediator and a target of IFN signals, STAT1 expression reports on, and determines IFN activity. Gene expression analyses of melanoma patient samples revealed varied levels of STAT1 expression, which highly correlated with expression of >700 genes. The ability of oncolytic viruses to exploit tumor-induced defects to antiviral responses suggests that oncolytic viruses may efficiently target a subset of melanomas, yet these should be defined. We modeled this scenario with murine B16F10 melanomas, immortalized skin fibroblasts as controls and a novel oncolytic virus, EHDV-TAU. In B16F10 cells, constitutive low expression of STAT1 and its target genes, which included intracellular pattern recognition receptors (PRRs), correlated with their inability to mount IFN-based antiviral responses upon EHDV-TAU challenge, and with potency of EHDV-TAU-induced oncolysis. This underexpression of interferon stimulated genes (ISGs) and PRRs, and the inability of EHDV-TAU to induce their expression, were reversed by epigenetic modifiers, suggesting epigenetic silencing as a basis for their underexpression. Despite their inability to mount IFN/STAT-based responses upon viral infection, EHDV-TAU infected B16F10 cells secreted immune-stimulatory chemokines. Accordingly, in vivo, EHDV-TAU enhanced intratumoral infiltration of cytotoxic T-cells and reduced growth of local and distant tumors. We propose that "STAT1 signatures" should guide melanoma virotherapy treatments, and that oncolytic viruses such as EHDV-TAU have the potential to exploit the cellular context of low-STAT1 tumors.

Safety and immunogenicity of 17DD attenuated yellow fever vaccine in howler monkeys (Alouatta spp.).

BACKGROUND: Alouatta spp. are highly susceptible to yellow fever (YF) infection and develop an often fatal disease. The threat posed by an outbreak started in 2016 leads us to investigate vaccination as a potential tool in preventing YF in non-human primates (NHP). METHODS: Susceptible howler monkeys were immunized with three different concentrations of the human Brazilian commercial YF17DD vaccine. Post-vaccination viremia/RNAemia, immunogenicity, and safety were characterized. RESULTS: The vaccine did not produce YF clinical manifestations in any of the NHPs. After immunization, all animals seroconverted demonstrating the ability of the YF vaccine to induce humoral response in Alouatta species. CONCLUSIONS: The present work has demonstrated the safe and immunogenic profile of the existing YF 17DD vaccine in howler monkeys. This knowledge may support further studies with other susceptible monkey species and provide a possible solution for controlling epizootics and preventing the devastation of endangered species.

Development and evaluation of recombinase polymerase amplification combined with lateral flow dipstick assays for co-detection of epizootic haemorrhagic disease virus and the Palyam serogroup virus.

BACKGROUND: Epizootic haemorrhagic disease virus (EHDV) and the Palyam serogroup viruses (PALV) have led to significant economic losses associated with livestock production globally. A rapid, sensitive and specific method for the detection of EHDV and PALV is critical for virus detection, monitoring, and successful control and elimination of related diseases. RESULTS: In the present study, a recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) assay for the co-detection of genome segment 1 (Seg-1) of EHDV and PALV was developed and evaluated. The analytical sensitivities of the established RPA-LFD assay in the detection of EHDV and PALV were 7.1 copies/µL and 6.8 copies/µL, respectively. No cross-reaction with other members of the genus Orbivirus, including African horse sickness virus, bluetongue virus, Guangxi orbivirus, Tibet orbivirus and Yunnan orbivirus was observed. The established RPA-LFD assay accurately detected 39 EHDV strains belonging to 5 serotypes and 29 PALV strains belonging to 3 serotypes. The trace back results of quantitative real-time polymerase chain reaction (qRT-PCR) and the established RPA-LFD assay on sentinel cattle were consistent. The coincidence rates of qRT-PCR and the established RPA-LFD assay in 56 blood samples from which EHDV or PALV had been isolated and 96 blood samples collected from cattle farms were more than 94.8 %. The results demonstrated that the established RPR-LFD assay is specific, sensitive and reliable, and could be applied in early clinical diagnosis of EHDV and PALV. CONCLUSIONS: This study highlights the development and application of the RPA-LFD assay in the co-detection of EHDV and PALV for the first time. The assay could be used as a potential optional rapid, reliable, sensitive and low-cost method for field diagnosis of EHDV and PALV.