The genetic diversity of Nipah virus across spatial scales.

BACKGROUND: Nipah virus (NiV), a highly lethal virus in humans, circulates in Pteropus bats throughout South and Southeast Asia. Difficulty in obtaining viral genomes from bats means we have a poor understanding of NiV diversity. METHODS: We develop phylogenetic approaches applied to the most comprehensive collection of genomes to date (N=257, 175 from bats, 73 from humans) from six countries over 22 years (1999-2020). We divide the four major NiV sublineages into 15 genetic clusters. Using Approximate Bayesian Computation fit to a spatial signature of viral diversity, we estimate the presence and the average size of genetic clusters per area. RESULTS: We find that, within any bat roost, there are an average of 2.4 co-circulating genetic clusters, rising to 5.5 clusters at areas of 1500-2000km2. We estimate that each genetic cluster occupies an average area of 1.3million km2 (95%CI: 0.6-2.3 million), with 14 clusters in an area of 100,000km2 (95%CI: 6-24). In the few sites in Bangladesh and Cambodia where genomic surveillance has been concentrated, we estimate that most clusters have been identified, but only ∼15% of overall NiV diversity has been uncovered. CONCLUSION: Our findings are consistent with entrenched co-circulation of distinct lineages, even within roosts, coupled with slow migration over larger spatial scales.

Seroprevalence and renal carriage of pathogenic Leptospira in livestock in Cotonou, Benin.

BACKGROUND: Leptospirosis is a zoonotic disease. It is particularly prevalent in tropical countries and has major consequences for human and animal health. In Benin, the disease's epidemiology remains poorly understood, especially in livestock, for which data are lacking. OBJECTIVES: To characterise Leptospira seroprevalence and locally circulating serogroups in livestock from Cotonou and to estimate the prevalence of Leptospira renal carriage in cattle. METHODS: We conducted a cross-sectional study in February 2020 during which livestock were sampled at an abattoir and in an impoverished city district. We analysed blood samples from 279 livestock animals (i.e. cattle, sheep, goats and pigs) using the microscopic agglutination test. Additionally, samples of renal tissue from 100 cattle underwent 16s rRNA (rrs) real-time PCR analysis. RESULTS: For the 131 cattle, 85 sheep, and 50 goats tested, seroprevalence was 18% (95% confidence interval [CI] [12%, 26%]), 9% (95% CI [4%, 17%] and 2% (95% CI [0%, 9%]), respectively, and most of the seropositive animals were associated with 1:100 titres. All 13 pigs were seronegative. Leptospira DNA was found in the renal tissue of 10% (95% CI [5%, 18%]) of the cattle tested (n = 100). Leptospira borgpetersenii was the main species present (n = 7), but Leptospira interrogans (n = 2) and Leptospira kirschneri (n = 1) were also detected. Various serogroups (Canicola, Grippotyphosa, Sejroe, Icterohaemorrhagiae, Pomona, Pyrogenes, Australis and Autumnalis) were detected using microscopic agglutination test without a clear predominance of any of them. CONCLUSIONS: These results suggest that abattoir workers and people living in close contact with livestock in poor urban areas are exposed to the risk of Leptospira infection.

Evisceration with implantation of an intrascleral silicone prosthesis in dogs: A retrospective study of 107 cases (2010-2019).

OBJECTIVE: To retrospectively analyze the preoperative clinical characteristics and surgical outcomes of evisceration with implantation of an intrascleral silicone prosthesis (EIISP) procedures in dogs and evaluate whether brachycephalic dogs are more prone than non-brachycephalic dogs to develop postoperative complications after EIISP. ANIMAL STUDIED: Ninety-One dogs (19 of which were brachycephalic) were included. PROCEDURES: Medical records from 2010 to 2019 were reviewed. Signalment, reason for EIISP, postoperative complications, follow-up time, and postoperative eye appearance were analyzed. RESULTS: The most frequently represented breeds were French Bulldog [11/91 (12%) dogs], Jack Russell Terrier [6/91 (7%)], and Shih Tzu [6/91 (7%)]. Brachycephalic dogs were statistically younger than non-brachycephalic dogs at the time of EIISP (p = 1.61 × 10-5 ). Uncontrolled glaucoma was the most common reason for EIISP in both groups. Short-term complications (from D0 to D15) seen in 7/91 (8%) dogs included epithelial corneal ulcers (n = 3), keratoconjunctivitis sicca (KCS) (n = 2), and prosthesis extrusion (n = 2). Long-term complications seen in 26/91 (29%) dogs included KCS (n = 11), epithelial corneal ulcers (n = 7), stromal ulcers (n = 3), entropion (n = 4), and prosthesis extrusion (n = 1). Extrusion of the prosthesis occurred twice in eyes that had undergone diode laser transscleral cyclophotocoagulation. The risk of postoperative complications was not significantly different between brachycephalic and non-brachycephalic dogs (p = .3). CONCLUSIONS: Brachycephalic status in dogs does not appear to influence the risk of complications from EIISP. Nevertheless, the present study is a reminder that EIISP focuses on esthetics, and considering the possible complications associated with it, it does not provide a benefit to the patient compared to enucleation.

Assessment of virus and Leptospira carriage in bats in France.

With over 1,400 species worldwide, bats represent the second largest order of mammals after rodents, and are known to host major zoonotic pathogens. Here, we estimate the presence of pathogens in autochthonous bat populations. First, we set out to check our samples for PCR amplification efficiency by assessing the occurrence of inhibited PCR reactions from different types of bat samples with amplifying the housekeeping gene ß-actin. Second, we investigated the presence of five targeted pathogens in a French bat population using PCR. We targeted viral RNA of Canine distemper virus, Alphacoronavirus, Lyssavirus, Rotavirus and bacterial Leptospira DNA. To do so, we screened for these viruses in bat faecal samples as well as in oropharyngeal swab samples. The presence of Leptospira was assessed in urine, kidney, lung and faecal samples. Results showed a frequency of inhibited reactions ranging from 5 to 60% of samples, varying according to the sample itself and also suspected to vary according to sampling method and the storage buffer solution used, demonstrating the importance of the sampling and storage on the probability of obtaining negative PCR results. For pathogen assessment, rotavirus and alphacoronavirus RNA were detected in Myotis myotis, Myotis daubentonii, Myotis emarginatus and Rhinolophus ferrumequinum bats. Rotaviruses were also detected in Barbastella barbastellus. The presence of alphacoronavirus also varied seasonally, with higher frequencies in late summer and October, suggesting that juveniles potentially play an important role in the dynamics of these viruses. Leptospira DNA was detected in M. myotis and M. daubentonii colonies. The 16S rRNA sequences obtained from Leptospira positive samples showed 100% genetic identity with L. borgpetersenii. Neither canine distemper virus nor lyssavirus RNA were detected in any of the tested samples. This study is the first to show the presence of Leptospira in autochthonous French bats in addition to coronavirus and rotavirus RNA previously reported in European autochthonous bats.

The genetic diversity of Nipah virus across spatial scales.

Nipah virus (NiV), a highly lethal virus in humans, circulates silently in Pteropus bats throughout South and Southeast Asia. Difficulty in obtaining genomes from bats means we have a poor understanding of NiV diversity, including how many lineages circulate within a roost and the spread of NiV over increasing spatial scales. Here we develop phylogenetic approaches applied to the most comprehensive collection of genomes to date (N=257, 175 from bats, 73 from humans) from six countries over 22 years (1999-2020). In Bangladesh, where most human infections occur, we find evidence of increased spillover risk from one of the two co-circulating sublineages. We divide the four major NiV sublineages into 15 genetic clusters (emerged 20-44 years ago). Within any bat roost, there are an average of 2.4 co-circulating genetic clusters, rising to 5.5 clusters at areas of 1,500-2,000 km2. Using Approximate Bayesian Computation fit to a spatial signature of viral diversity, we estimate that each genetic cluster occupies an average area of 1.3 million km2 (95%CI: 0.6-2.3 million), with 14 clusters in an area of 100,000 km2 (95%CI: 6-24). In the few sites in Bangladesh and Cambodia where genomic surveillance has been concentrated, we estimate that most of the genetic clusters have been identified, but only ~15% of overall NiV diversity has been uncovered. Our findings are consistent with entrenched co-circulation of distinct lineages, even within individual roosts, coupled with slow migration over larger spatial scales.

Foraging and mating behaviors of Hypsignathus monstrosus at the bat-human interface in a central African rainforest.

Studying wildlife space use in human-modified environments contributes to characterize wildlife-human interactions to assess potential risks of zoonotic-pathogens transmission, and to pinpoint conservation issues. In central African rainforests with human dwelling and activities, we conducted a telemetry study on a group of males of Hypsignathus monstrosus, a lek-mating fruit bat identified as a potential maintenance host for Ebola virus. During a lekking season in 2020, we investigated the foraging-habitat selection and the individual nighttime space use during both mating and foraging activities close to villages and their surrounding agricultural landscape. At night, marked individuals strongly selected agricultural lands and more generally areas near watercourses to forage, where they spent more time compared to forest ones. Furthermore, the probability and duration of the presence of bats in the lek during nighttime decreased with the distance to their roost site but remained relatively high within a 10 km radius. Individuals adjusted foraging behaviors according to mating activity by reducing both the overall time spent in foraging areas and the number of forest areas used to forage when they spent more time in the lek. Finally, the probability of a bat revisiting a foraging area in the following 48 hours increased with the previous time spent in that foraging area. These behaviors occurring close to or in human-modified habitats can trigger direct and indirect bat-human contacts, which could thus facilitate pathogen transmission such as Ebola virus.

Assessment of inter-observer agreement among experienced and inexperienced observers and an automated device for dipstick urinalysis in dogs and cats.

Standard visual urine dipstick analysis (UDA) is performed routinely in veterinary medicine; results can be influenced by both the operator and the method. We evaluated the agreement of results for canine and feline urine samples analyzed using a 10-patch dipstick (Multistix10SG; Siemens), both visually under double-anonymized conditions by students and a laboratory technician, and with an automated device (AD; Clinitek Status, Siemens). The mean concordance for semiquantitative urinalysis results between students and the technician and between students and the AD was fair (κ0.21-0.40) in dogs and cats; concordance was moderate between the technician and the AD (κ0.41-0.60) in dogs and good (κ0.61-0.80) in cats. For pH, the mean concordance between students and the technician and between the technician and the AD was good (ρ0.80-0.92) in dogs and cats; concordance was good between students and the AD (ρ0.80-0.92) in dogs and moderate (ρ0.59-0.79) in cats. Repeatability was higher (p < 0.001) for the technician and the AD than for a student. We found good agreement between UDA performed by an experienced operator and an AD in dogs and cats but found low reproducibility and low repeatability for urinalysis performed by an inexperienced operator.

Effect of an ear cleaner instillation containing lipacids in a model of re-acidification of the external auditory canal in dogs.

BACKGROUND: In humans, the acidic pH of the ear canal plays a protective role against infection and a change towards alkalinity of the external auditory canal (EAC) is a local factor in the progression of acute to chronic otitis externa (OE). The use of acidic preparations alone for treatment of OE without concurrent antibiotic use is well-documented in humans. In dogs, only one study has investigated the EAC pH in healthy dogs and in dogs with OE, and investigations to understand the role of EAC pH in the pathogenesis of canine OE are lacking. HYPOTHESIS/OBJECTIVES: To obtain physiological EAC pH values in beagle dogs. To develop a model of re-acidification of the EAC in dogs and to investigate how an acidic solution may accelerate the return to a physiological pH. ANIMALS: Ten healthy beagle dogs in a laboratory setting. MATERIALS AND METHODS: A model of re-acidification of the EAC was developed by instillation of a pH 10.1 phosphate-buffered saline (PBS) solution and the subsequent acidic effect of an ear cleaner containing lipacids was evaluated in this model. RESULTS: Mean physiological EAC pH was 6.12 (± 0.36). EAC re-acidification took up to 9 h in this model. Mean pH values dropped immediately to 6.38 (± 0.27) on ears treated with an acidic ear cleaner. No abrupt drop was observed of the mean pH values for the control ears. CONCLUSION AND CLINICAL IMPORTANCE: This study confirms that physiological EAC pH in dogs is acidic. This model of re-acidification of the EAC pH allows investigations on acidic properties of topical ear products in healthy ears.

Contexte - Chez l'homme, le pH acide du conduit auditif joue un rôle protecteur contre l'infection et l'évolution vers l'alcalinité du conduit auditif externe (CAE) et est un facteur local de progression de l'otite externe (OE) aiguë à chronique. L'utilisation de préparations acides seules pour le traitement de l'OE sans utilisation concomitante d'antibiotiques est bien documentée chez l'homme. Chez les chiens, une seule étude a étudié le pH du CAE chez les chiens en bonne santé et chez les chiens atteints d'OE, et les recherches pour comprendre le rôle du pH du CAE dans la pathogenèse de l'OE canine font défaut. Hypothèses/objectifs - Obtenir des valeurs physiologiques de pH du CAE chez des chiens beagle. Développer un modèle de réacidification du CAE chez le chien et étudier comment une solution acide peut accélérer le retour à un pH physiologique. Animaux - Dix chiens beagle de laboratoire en bonne santé. Matériels et méthodes - Un modèle de réacidification du CAE a été développé par instillation d'une solution saline tamponnée (PBS) à pH 10,1 et l'effet acide ultérieur d'un nettoyant pour oreilles contenant des lipacides a été évalué dans ce modèle. Résultats - Le pH physiologique moyen du CAE était de 6,12 (± 0,36). La réacidification du CAE a pris jusqu'à 9 h dans ce modèle. Les valeurs moyennes du pH chutent immédiatement à 6,38 (± 0,27) sur les oreilles traitées avec un nettoyant auriculaire acide. Aucune chute brutale n'a été observée des valeurs moyennes de pH pour les oreilles témoins. Conclusion et importance clinique - Cette étude confirme que le pH physiologique du CAE chez le chien est acide. Ce modèle de réacidification du pH du CAE permet des investigations sur les propriétés acides des produits topiques auriculaires dans des oreilles saines.

Introducción; en humanos, el pH ácido del canal auditivo juega un papel protector contra la infección y un cambio hacia la alcalinidad del canal auditivo externo (EAC) es un factor local en la progresión de la otitis externa (OE) aguda a crónica. El uso de preparaciones ácidas solas para el tratamiento de la OE sin el uso concomitante de antibióticos está bien documentado en humanos. En perros, solo un estudio ha investigado el pH de EAC en perros sanos y en perros con OE, y faltan investigaciones para comprender el papel del pH de EAC en la patogenia de la OE canina. Hipótesis/objetivos - Obtener valores de pH fisiológico de EAC en perros beagle. Desarrollar un modelo de reacidificación del EAC en perros e investigar cómo una solución ácida puede acelerar el retorno a un pH fisiológico. Animales- diez perros beagle sanos en un laboratorio. Materiales y métodos- se desarrolló un modelo de reacidificación del EAC mediante la instilación de una solución salina tamponada con fosfato (PBS) de pH 10,1 y se evaluó en este modelo el efecto ácido subsiguiente de un limpiador de oídos que contenía lípidos. Resultados - El pH fisiológico medio del EAC fue de 6,12 (± 0,36). La reacidificación de EAC tomó hasta 9 h en este modelo. Los valores medios de pH cayeron inmediatamente a 6,38 (± 0,27) en los oídos tratados con un limpiador de oídos ácido. No se observó una caída abrupta de los valores medios de pH para los oíds de control. Conclusión e importancia clínica- este estudio confirma que el pH fisiológico de EAC en perros es ácido. Este modelo de reacidificación del pH de EAC permite realizar investigaciones sobre las propiedades ácidas de los productos tópicos para el oído en oídos sanos.

Contexto - Em humanos, o pH ácido do conduto auditivo exerce uma grande função protetora contra infecções e a alcalinização do conduto auditivo externo (CAE) é um fator local de progressão de otite externa (OE) aguda para crônica. A utilização unicamente de formulações ácidas para o tratamento de OE sem a utilização concomitante de antibióticos é bem documentada em humanos. Em cães, apenas um estudo investigou o pH do CAE em cães saudáveis com OE, e são escassas as pesquisas investigando o papel do pH do CAE na patogênese da OE. Hipótese/objetivos - Se obter os valores fisiológicos do pH dos CAEs de cães Beagle. Desenvolver um modelo de re-acidificação do CAE em cães e investigar como uma solução ácida pode acelerar o retorno ao pH fisiológico original. Animais - Dez cães Beagle saudáveis de laboratório. Materiais e Métodos - Um modelo de re-acidificação do CAE foi desenvolvido por instilação de solução salina tamponada com fosfato (PBS) com um pH 10,1 e o efeito acidificante subsequente de um limpador de ouvido contendo ácidos lipídicos foi avaliado neste modelo. Resultados - O pH fisiológico médio do CAE foi de 6,12 (± 0,36). A re-acidificação do CAE levou até 9h neste modelo. Os valores médios de pH caíram imediatamente para 6,38 (± 0,27) nas orelhas tratadas com um limpador otológico ácido. Não foi observada queda abrupta dos valores médios de pH para as orelhas controle. Conclusão e importância clínica - Este estudo confirmou que o pH fisiológico do CAE de cães é ácido. Este modelo de re-acidificação do pH do CAE permite investigações sobre as propriedades acidificantes de produtos otológicos tópicos para orelhas saudáveis.

Quantification and characterisation of commensal wild birds and their interactions with domestic ducks on a free-range farm in southwest France.

The role of commensal birds in the epidemiology of pathogens in poultry farms remains unclear. Our study aimed to identify potential key species for interactions with domestic ducks on one free-range duck farm in southwest France. Methods combined direct individual observations on duck outdoor foraging areas, network analysis, and general linear mixed models of abundances. Results showed a wide diversity of wild bird species visiting foraging areas, heavily dominated in frequency by White wagtails (Motacilla alba) and Sparrows (Passer domesticus and Passer montanus). These also were the only species seen entering duck premises or perching on drinkers in the presence of ducks. Moreover, White wagtails were the species most frequently observed on the ground and in close proximity to ducks. Network analysis suggested the role of White wagtails and Sparrows in linking ducks to other wild birds on the farm. The abundance of White wagtails was positively associated with open vegetation, with the presence of ducks and particularly in the afternoon, while the abundance of Sparrows was positively associated only with the fall-winter season. By precisely characterising interactions, the study was able to identify few wild bird species which should be prioritized in infectious investigations at the interface with poultry.

Childhood encephalitis in the Greater Mekong region (the SouthEast Asia Encephalitis Project): a multicentre prospective study.

BACKGROUND: Encephalitis is a worldwide public health issue, with a substantially high burden among children in southeast Asia. We aimed to determine the causes of encephalitis in children admitted to hospitals across the Greater Mekong region by implementing a comprehensive state-of-the-art diagnostic procedure harmonised across all centres, and identifying clinical characteristics related to patients' conditions. METHODS: In this multicentre, observational, prospective study of childhood encephalitis, four referral hospitals in Cambodia, Vietnam, Laos, and Myanmar recruited children (aged 28 days to 16 years) who presented with altered mental status lasting more than 24 h and two of the following minor criteria: fever (within the 72 h before or after presentation), one or more generalised or partial seizures (excluding febrile seizures), a new-onset focal neurological deficit, cerebrospinal fluid (CSF) white blood cell count of 5 per mL or higher, or brain imaging (CT or MRI) suggestive of lesions of encephalitis. Comprehensive diagnostic procedures were harmonised across all centres, with first-line testing was done on samples taken at inclusion and results delivered within 24 h of inclusion for main treatable causes of disease and second-line testing was done thereafter for mostly non-treatable causes. An independent expert medical panel reviewed the charts and attribution of causes of all the included children. Using multivariate analyses, we assessed risk factors associated with unfavourable outcomes (ie, severe neurological sequelae and death) at discharge using data from baseline and day 2 after inclusion. This study is registered with ClinicalTrials.gov, NCT04089436, and is now complete. FINDINGS: Between July 28, 2014, and Dec 31, 2017, 664 children with encephalitis were enrolled. Median age was 4·3 years (1·8-8·8), 295 (44%) children were female, and 369 (56%) were male. A confirmed or probable cause of encephalitis was identified in 425 (64%) patients: 216 (33%) of 664 cases were due to Japanese encephalitis virus, 27 (4%) were due to dengue virus, 26 (4%) were due to influenza virus, 24 (4%) were due to herpes simplex virus 1, 18 (3%) were due to Mycobacterium tuberculosis, 17 (3%) were due to Streptococcus pneumoniae, 17 (3%) were due to enterovirus A71, 74 (9%) were due to other pathogens, and six (1%) were due to autoimmune encephalitis. Diagnosis was made within 24 h of admission to hospital for 83 (13%) of 664 children. 119 (18%) children had treatable conditions and 276 (42%) had conditions that could have been preventable by vaccination. At time of discharge, 153 (23%) of 664 children had severe neurological sequelae and 83 (13%) had died. In multivariate analyses, risk factors for unfavourable outcome were diagnosis of M tuberculosis infection upon admission (odds ratio 3·23 [95% CI 1·04-10·03]), coma on day 2 (2·90 [1·78-4·72]), supplementary oxygen requirement (1·89 [1·25-2·86]), and more than 1 week duration between symptom onset and admission to hospital (3·03 [1·68-5·48]). At 1 year after inclusion, of 432 children who were discharged alive from hospital with follow-up data, 24 (5%) had died, 129 (30%) had neurological sequelae, and 279 (65%) had completely recovered. INTERPRETATION: In southeast Asia, most causes of childhood encephalitis are either preventable or treatable, with Japanese encephalitis virus being the most common cause. We provide crucial information that could guide public health policy to improve diagnostic, vaccination, and early therapeutic guidelines on childhood encephalitis in the Greater Mekong region. FUNDING: Institut Pasteur, Institut Pasteur International Network, Fondation Merieux, Aviesan Sud, INSERM, Wellcome Trust, Institut de Recherche pour le Développement (IRD), and Fondation Total.

Longitudinal Survey of Coronavirus Circulation and Diversity in Insectivorous Bat Colonies in Zimbabwe.

BACKGROUND: Studies have linked bats to outbreaks of viral diseases in human populations such as SARS-CoV-1 and MERS-CoV and the ongoing SARS-CoV-2 pandemic. METHODS: We carried out a longitudinal survey from August 2020 to July 2021 at two sites in Zimbabwe with bat-human interactions: Magweto cave and Chirundu farm. A total of 1732 and 1866 individual bat fecal samples were collected, respectively. Coronaviruses and bat species were amplified using PCR systems. RESULTS: Analysis of the coronavirus sequences revealed a high genetic diversity, and we identified different sub-viral groups in the Alphacoronavirus and Betacoronavirus genus. The established sub-viral groups fell within the described Alphacoronavirus sub-genera: Decacovirus, Duvinacovirus, Rhinacovirus, Setracovirus and Minunacovirus and for Betacoronavirus sub-genera: Sarbecoviruses, Merbecovirus and Hibecovirus. Our results showed an overall proportion for CoV positive PCR tests of 23.7% at Chirundu site and 16.5% and 38.9% at Magweto site for insectivorous bats and Macronycteris gigas, respectively. CONCLUSIONS: The higher risk of bat coronavirus exposure for humans was found in December to March in relation to higher viral shedding peaks of coronaviruses in the parturition, lactation and weaning months of the bat populations at both sites. We also highlight the need to further document viral infectious risk in human/domestic animal populations surrounding bat habitats in Zimbabwe.

Circulating serogroups of Leptospira in swine from a 7-year study in France (2011-2017).

BACKGROUND: Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira and is responsible for significant economic porcine livestock losses. Knowledge of Leptospira serogroups and their distributions is important for evaluation of the relevance of leptospirosis management measures, including use of the prophylactic vaccine that was recently made available in France. A retrospective study was conducted to determine the relationships between different circulating Leptospira serogroups. Pigs from across France presenting clinical signs suggestive of leptospirosis were tested with the microagglutination test (MAT) between 2011 and 2017. We used weighted averages to determine serogroup distributions according to MAT results and considering cross-reactions. RESULTS: A total of 19,395 pig sera, mostly from Brittany, were tested, and 22.7% were found to be positive for at least one Leptospira serogroup. Analysis of the 4,346 seropositive results for which the putative infective serogroup could be defined, revealed that two out of ten serogroups were much more frequent than the others: Australis (48.5%) and Icterohaemorrhagiae (38.2%). Other serogroups, including Autumnalis, Panama, Ballum, Tarassovi, Sejroe, Grippotyphosa, Bataviae, and Pomona, were less common. CONCLUSIONS: Although diagnostic laboratory data cannot be extrapolated to infer the distribution of Leptospira serogroups at the nationwide scale in France, the analysis of such data can provide an overview of the relationship between circulating Leptospira serogroups in space and time. During the last decade, protection against the serogroups Australis and Icterohaemorrhagiae would have prevented most of the clinical porcine leptospirosis cases in the large number of farms that we studied. In the future, epidemiological information related to circulating Leptospira serogroups should be extracted from data with a standardized approach for use in nationwide or international surveillance and prophylactic strategy support.

Spatial-temporal patterns and risk factors for human leptospirosis in Thailand, 2012-2018.

Leptospirosis is a globally important zoonotic disease. The disease is particularly important in tropical and subtropical countries. Infections in humans can be caused by exposure to infected animals or contaminated soil or water, which are suitable for Leptospira. To explore the cluster area, the Global Moran's I index was calculated for incidences per 100,000 population at the province level during 2012-2018, using the monthly and annual data. The high-risk and low-risk provinces were identified using the local indicators of spatial association (LISA). The risk factors for leptospirosis were evaluated using a generalized linear mixed model (GLMM) with zero-inflation. We also added spatial and temporal correlation terms to take into account the spatial and temporal structures. The Global Moran's I index showed significant positive values. It did not demonstrate a random distribution throughout the period of study. The high-risk provinces were almost all in the lower north-east and south parts of Thailand. For yearly reported cases, the significant risk factors from the final best-fitted model were population density, elevation, and primary rice crop arable areas. Interestingly, our study showed that leptospirosis cases were associated with large areas of rice production but were less prevalent in areas of high rice productivity. For monthly reported cases, the model using temperature range was found to be a better fit than using percentage of flooded area. The significant risk factors from the model using temperature range were temporal correlation, average soil moisture, normalized difference vegetation index, and temperature range. Temperature range, which has strongly negative correlation to percentage of flooded area was a significant risk factor for monthly data. Flood exposure controls should be used to reduce the risk of leptospirosis infection. These results could be used to develop a leptospirosis warning system to support public health organizations in Thailand.

Dynamics of Antibodies to Ebolaviruses in an Eidolon helvum Bat Colony in Cameroon.

The ecology of ebolaviruses is still poorly understood and the role of bats in outbreaks needs to be further clarified. Straw-colored fruit bats (Eidolon helvum) are the most common fruit bats in Africa and antibodies to ebolaviruses have been documented in this species. Between December 2018 and November 2019, samples were collected at approximately monthly intervals in roosting and feeding sites from 820 bats from an Eidolon helvum colony. Dried blood spots (DBS) were tested for antibodies to Zaire, Sudan, and Bundibugyo ebolaviruses. The proportion of samples reactive with GP antigens increased significantly with age from 0-9/220 (0-4.1%) in juveniles to 26-158/225 (11.6-70.2%) in immature adults and 10-225/372 (2.7-60.5%) in adult bats. Antibody responses were lower in lactating females. Viral RNA was not detected in 456 swab samples collected from 152 juvenile and 214 immature adult bats. Overall, our study shows that antibody levels increase in young bats suggesting that seroconversion to Ebola or related viruses occurs in older juvenile and immature adult bats. Multiple year monitoring would be needed to confirm this trend. Knowledge of the periods of the year with the highest risk of Ebolavirus circulation can guide the implementation of strategies to mitigate spill-over events.

Detection and genetic diversity of Mopeia virus in Mastomys natalensis from different habitats in the Limpopo National Park, Mozambique.

Mammarenaviruses have been a growing concern for public health in Africa since the 1970s when Lassa virus cases in humans were first described in west Africa. In southern Africa, a single outbreak of Lujo virus was reported to date in South Africa in 2008 with a case fatality rate of 80%. The natural reservoir of Lassa virus is Mastomys natalensis while for the Lujo virus the natural host has yet to be identified. Mopeia virus was described for the first time in M. natalensis in the central Mozambique in 1977 but few studies have been conducted in the region. In this study, rodents were trapped between March and November 2019in villages, croplands fields and mopane woodland forest. The aim was to assess the potential circulation and to evaluate the genetic diversity of mammarenaviruses in M. natalensis trapped in the Limpopo National Park and its buffer zone in Massingir district, Mozambique. A total of 534 M. natalensis were screened by RT-PCR and the overall proportion of positive individuals was 16.9%. No significant differences were detected between the sampled habitats (χ2 = 0.018; DF = 1; p = 0.893). The Mopeia virus (bootstrap value 91%) was the Mammarenavirus circulating in the study area sites, forming a specific sub-clade with eight different sub-clusters. We concluded that Mopeia virus circulates in all habitats investigated and it forms a different sub-clade to the one reported in central Mozambique in 1977.

Genetic identification of bat species for pathogen surveillance across France.

With more than 1400 chiropteran species identified to date, bats comprise one-fifth of all mammalian species worldwide. Many studies have associated viral zoonoses with 45 different species of bats in the EU, which cluster within 5 families of bats. For example, the Serotine bats are infected by European Bat 1 Lyssavirus throughout Europe while Myotis bats are shown infected by coronavirus, herpesvirus and paramyxovirus. Correct host species identification is important to increase our knowledge of the ecology and evolutionary pattern of bat viruses in the EU. Bat species identification is commonly determined using morphological keys. Morphological determination of bat species from bat carcasses can be limited in some cases, due to the state of decomposition or nearly indistinguishable morphological features in juvenile bats and can lead to misidentifications. The overall objective of our study was to identify insectivorous bat species using molecular biology tools with the amplification of the partial cytochrome b gene of mitochondrial DNA. Two types of samples were tested in this study, bat wing punches and bat faeces. A total of 163 bat wing punches representing 22 species, and 31 faecal pellets representing 7 species were included in the study. From the 163 bat wing punches tested, a total of 159 were genetically identified from amplification of the partial cyt b gene. All 31 faecal pellets were genetically identified based on the cyt b gene. A comparison between morphological and genetic determination showed 21 misidentifications from the 163 wing punches, representing ~12.5% of misidentifications of morphological determination compared with the genetic method, across 11 species. In addition, genetic determination allowed the identification of 24 out of 25 morphologically non-determined bat samples. Our findings demonstrate the importance of a genetic approach as an efficient and reliable method to identify bat species precisely.

Longitudinal monitoring in Cambodia suggests higher circulation of alpha and betacoronaviruses in juvenile and immature bats of three species.

Recent studies suggest that coronaviruses circulate widely in Southeast Asian bat species and that the progenitors of the SARS-Cov-2 virus could have originated in rhinolophid bats in the region. Our objective was to assess the diversity and circulation patterns of coronavirus in several bat species in Southeast Asia. We undertook monthly live-capture sessions and sampling in Cambodia over 17 months to cover all phases of the annual reproduction cycle of bats and test specifically the association between their age and CoV infection status. We additionally examined current information on the reproductive phenology of Rhinolophus and other bat species presently known to occur in mainland southeast China, Vietnam, Laos and Cambodia. Results from our longitudinal monitoring (573 bats belonging to 8 species) showed an overall proportion of positive PCR tests for CoV of 4.2% (24/573) in cave-dwelling bats from Kampot and 4.75% (22/463) in flying-foxes from Kandal. Phylogenetic analysis showed that the PCR amplicon sequences of CoVs (n = 46) obtained clustered in Alphacoronavirus and Betacoronavirus. Interestingly, Hipposideros larvatus sensu lato harbored viruses from both genera. Our results suggest an association between positive detections of coronaviruses and juvenile and immature bats in Cambodia (OR = 3.24 [1.46-7.76], p = 0.005). Since the limited data presently available from literature review indicates that reproduction is largely synchronized among rhinolophid and hipposiderid bats in our study region, particularly in its more seasonal portions (above 16° N), this may lead to seasonal patterns in CoV circulation. Overall, our study suggests that surveillance of CoV in insectivorous bat species in Southeast Asia, including SARS-CoV-related coronaviruses in rhinolophid bats, could be targeted from June to October for species exhibiting high proportions of juveniles and immatures during these months. It also highlights the need to develop long-term longitudinal surveys of bats and improve our understanding of their ecology in the region, for both biodiversity conservation and public health reasons.

Mapping of Ebola virus spillover: Suitability and seasonal variability at the landscape scale.

The unexpected Ebola virus outbreak in West Africa in 2014 involving the Zaire ebolavirus made clear that other regions outside Central Africa, its previously documented niche, were at risk of future epidemics. The complex transmission cycle and a lack of epidemiological data make mapping areas at risk of the disease challenging. We used a Geographic Information System-based multicriteria evaluation (GIS-MCE), a knowledge-based approach, to identify areas suitable for Ebola virus spillover to humans in regions of Guinea, Congo and Gabon where Ebola viruses already emerged. We identified environmental, climatic and anthropogenic risk factors and potential hosts from a literature review. Geographical data layers, representing risk factors, were combined to produce suitability maps of Ebola virus spillover at the landscape scale. Our maps show high spatial and temporal variability in the suitability for Ebola virus spillover at a fine regional scale. Reported spillover events fell in areas of intermediate to high suitability in our maps, and a sensitivity analysis showed that the maps produced were robust. There are still important gaps in our knowledge about what factors are associated with the risk of Ebola virus spillover. As more information becomes available, maps produced using the GIS-MCE approach can be easily updated to improve surveillance and the prevention of future outbreaks.

Agro-Environmental Determinants of Leptospirosis: A Retrospective Spatiotemporal Analysis (2004-2014) in Mahasarakham Province (Thailand).

Leptospirosis has been recognized as a major public health concern in Thailand following dramatic outbreaks. We analyzed human leptospirosis incidence between 2004 and 2014 in Mahasarakham province, Northeastern Thailand, in order to identify the agronomical and environmental factors likely to explain incidence at the level of 133 sub-districts and 1982 villages of the province. We performed general additive modeling (GAM) in order to take the spatial-temporal epidemiological dynamics into account. The results of GAM analyses showed that the average slope, population size, pig density, cow density and flood cover were significantly associated with leptospirosis occurrence in a district. Our results stress the importance of livestock favoring leptospirosis transmission to humans and suggest that prevention and control of leptospirosis need strong intersectoral collaboration between the public health, the livestock department and local communities. More specifically, such collaboration should integrate leptospirosis surveillance in both public and animal health for a better control of diseases in livestock while promoting public health prevention as encouraged by the One Health approach.

Formula for the estimation of urine osmolality in healthy cats.

A simple and intuitive formula for the estimation of urine osmolality (Uosm) using the measured concentrations of major active urine osmolytes over a wide range of urine dilutions was proposed in healthy cats. Sixty-three urine samples were retrieved using ultrasound-guided cystocentesis from sixteen healthy cats under 5 years of age receiving intravenous infusion over a period of 24 h. Samples were collected at baseline (T0), T2, T6, T12, and T24. Urine osmolality was measured using a freezing-point osmometer, and the concentrations of osmolytes (urea, sodium, glucose, and potassium) were evaluated. A simple linear regression model for a clinical use was selected, and the agreement between the calculated and actual urine osmolalities was assessed. Urinary concentrations of urea, sodium and glucose were the three variables included in the model with the lowest AICC. Urine osmolality can be predicted accurately and precisely using urine urea, sodium and glucose with the following equation: Uosm = 1.25 × urea (mmol/l) or 20.87 × urea (g/l) + 1.1 × sodium (mmol/l) + 67 × glucose (mmol/l) or 3.72 × glucose (mg/dl). The concordance correlation coefficient for repeated measures between the actual and the calculated urine osmolality was extremely close to 1, which supported a high agreement: 0.996 (CI 95%: [0.993; 0.998]). In a population of healthy cats, urine osmolality can be predicted accurately and precisely using urinary urea, sodium and glucose concentrations. Similar formulae could potentially be established to help the clinician in pathological situations.