We investigated the presence of hepatitis E virus (HEV) in pigs at the moment of slaughter in Spain in years 2015 and 2017. A total of 1786 caecal content, liver, and serum samples from animals at slaughterhouses were tested by reverse transcription real-time quantitative PCR (RT-qPCR) and anti-HEV antibodies were evaluated in 623 serum samples by an ELISA test. The overall seroprevalence obtained was 70.9 %. A total of 398 RT-qPCR positive samples were identified in caecal content (26.8 %; 156/583), serum (21.8 %; 136/623) and liver (18.3 %; 106/580). A total of 32 RT-qPCR positive samples were genotyped; 3f (84.4 %) and the 3c (9.4 %) being the most prevalent subgenotypes. This is the first report on detection of HEV in pigs at the moment of slaughter with a Spain nation-wide representation. The data show a large high seroprevalence (70.9 %) in pigs, while the presence of the virus (HEV RNA) was significantly lower. HEV RNA detection varied markedly between matrices, with caecal samples showing higher positivity (30-50 %) than serum (5-25 %); both simple and interaction GEE models confirmed strong effects of sample type and its interaction with year on prevalence estimates. However, the percentage of positive liver samples (18.3 %) and the concurrence between the HEV 3 subtypes identified (3f, 3m and 3c) and those identified in human patients in Spain, underscores the possibility of foodborne zoonosis. It can represent a real risk for consumers if pork products are not cooked adequately. A holistic One-Health approach, including a better understanding of HEV prevalence in the swine population, would allow implementation of control measures in the meat chain to mitigate the main transmission routes for humans.

As wild ungulates, including European bison, increasingly share habitats with livestock, surveillance of infectious zoonotic agents in their populations is essential for both wildlife and public health. This study aimed to screen for selected zoonotic pathogens in European bison from Poland. Samples (blood, ticks, and spleen) were collected from 86 animals. Serum was used for serological testing using commercial ELISA kits for Borrelia burgdorferi sensu lato, Brucella spp., and hepatitis E virus (HEV); ticks were analysed by real-time PCR targeting B. burgdorferi s.l., Anaplasma phagocytophilum, and Brucella spp., and spleen samples from Brucella-seropositive animals were cultured. Serological analysis revealed that 53.9% of European bison were seropositive for B. burgdorferi s.l., while 25.3% showed seroreactivity against Brucella spp.; however, these findings were not supported by molecular or culture confirmation, suggesting possible non-specific reactions or past exposure. No serum samples were positive for HEV antibodies, and no Brucella spp. were isolated from spleen samples. Molecular analysis of ticks detected B. burgdorferi s.l. DNA in 4.8% of samples and sequencing confirmed Borrelia garinii in one case. In contrast, A. phagocytophilum DNA was detected in 59.0% of ticks. No ticks tested positive for Brucella DNA. These findings indicate substantial exposure of European bison to tick-borne pathogens, particularly B. burgdorferi s.l. and A. phagocytophilum. However, Brucella seropositivity should be interpreted with caution due to the lack of molecular or culture confirmation.

Wild birds are relevant reservoirs and sentinels for zoonotic pathogens such as Chlamydia psittaci and West Nile virus (WNV), both of which can affect animal and public health. Wildlife rehabilitation centers (WRCs) offer unique opportunities for passive surveillance of emerging and re-emerging infectious diseases, particularly in urban and peri-urban settings. From 2013 to 2022, a total of 1024 bird samples were collected upon admission to WRCs in the Community of Madrid, Spain. Oropharyngeal and cloacal swabs, as well as tissue samples, were tested using real-time PCR targeting the ompA gene of C. psittaci and the 3'NC region of WNV. One sample tested positive for C. psittaci by real-time PCR in 2021, yielding a positivity rate of 0.22% (95% CI: 0.01-1.19). No positive cases were detected during the remaining years of the study. All samples tested negative for WNV over the nine-year period. The low detection rate suggests limited circulation of these pathogens among wild birds in central Spain, though it may partly reflect the variability inherent to passive surveillance and sample-type heterogeneity. However, continued surveillance is warranted, especially in high-risk avian species and personnel occupationally exposed in avian rehabilitation facilities using expanded sample sizes and complementary diagnostic tools. Extending monitoring beyond the typical vector season and increasing testing of sensitive tissues, particularly for WNV, may further enhance detection sensitivity and strengthen early-warning capacity. These efforts are essential to improve early detection and risk assessment within a One Health framework.

Brucellosis is a sanitary and economically relevant disease affecting humans, livestock, and wildlife. Ticks have been suggested as vectors, long-term carriers, and amplifiers of Brucella. In this study, ticks from wildlife ungulate hosts living in hunting reserves of a central region of Spain were collected during a 6-year period, pooled, and screened for Brucella spp. by PCR. Aiming to correlate Brucella spp. DNA presence in ticks with Brucella spp. infections in wildlife ungulate hosts, liver samples from deceased wildlife ungulates coming from the hunting reserves showing a positive result for Brucella in ticks were tested using a commercial ELISA. In total, 229 tick pools from wild boar (Sus scrofa, n = 176; 76.8%, 95% CI 70.9%-81.8%), red deer (Cervus elaphus, n = 40; 17.4%, 95% CI 13.1%-22.9%), mouflon (Ovis orientalis musimon, n = 7; 3.06%, 95% CI 1.49%-6.17%), and fallow deer (Dama dama, n = 6; 2.62%, 95% CI 1.21%-5.60%) were analyzed. PCR results showed that 3.93% (95% CI 2.08%-7.30%) tick pools (9/229) from 16.6% hunting reserves (7/41) screened yielded a positive PCR result for Brucella. All positive ticks were Dermacentor (Dermacentor marginatus or Dermacentor reticulatus) collected from wild boar. Ticks collected from wild boars were positive to Brucella in a relative percentage of 5.10% (95% CI = 1.61-11.4) in 2018 and of 7.59% (95% CI = 2.79-15.6) in 2021 (6-year prevalence of 5.17%, 9/176). ELISA showed positive results in three wild boars coming from two out of seven hunting reserves (28.5%) with a positive PCR for Brucella in ticks. To conclude, Brucella spp. DNA can be detected in Dermacentor ticks parasitizing wild boars living in hunting reserves harboring Brucella spp.-seropositive wild boars. This study provides evidence that the contribution of arthropod vectors should be considered in the epidemiology of brucellosis in wildlife.

Porcine brucellosis, caused by Brucella suis (B. suis), is a notifiable disease causing significant economic losses in production systems. Most infected pigs may act as carriers and shed B. suis even if asymptomatic. This can contribute to environmental persistence, thus hindering control efforts. Here, the environment and the offspring were investigated during and after a B. suis outbreak at a sow breeding farm. The diagnosis of B. suis in sows (n = 1,140) was performed by culture and polymerase chain reaction (PCR) from vaginal swabs, indirect enzyme-linked immunosorbent assay (I-ELISA) from sera, and brucellin skin test (BST). B. suis diagnosis in post-weaning pigs (n = 899) was performed by I-ELISA in sera and BST. The environmental surveillance programme was implemented by placing gauze sponges (n = 175) pre-hydrated in a surfactant and inactivating liquid for Brucella DNA detection by PCR in different farm areas. Our results showed that the offspring of infected sows reacted to in vivo techniques for B. suis. Furthermore, the offspring born during the outbreak displayed higher seropositivity (I-ELISA) and reactivity (BST) than those pigs born after. Brucella DNA was detected in pregnant sow areas, boxes, boots, and post-weaning pig areas. In addition, Brucella DNA environmental detection was higher during the B. suis outbreak than the post B. suis outbreak. The environmental approach has proven to be a simple, practical, valuable, and safe method to detect and monitor B. suis. These results suggest a role of the environment and the offspring that should be considered in porcine brucellosis surveillance and control programmes.

For the aim of genotypic fingerprinting of Brucella melitensis bv3 isolated from different ruminant species in Kafrelsheikh governorate, Egypt, a multilocus variable-number tandem-repeat analysis (MLVA 16) has been approached. The MLVA 16 was performed on 41 B. melitensis bv3 isolates identified by bacteriological and molecular techniques. Thirty-one isolates originated from the preferential host (28 sheep and three goats), and ten isolates from atypical hosts (nine cattle and one buffalo). Recovering the same genotype in two different animal species suggests cross-species adaptation of B. melitensis bv3 to different atypical ruminant species in Egypt. Furthermore, the isolation of B. melitensis from aborted cows after the entry of a replacement cow from an unknown brucellosis status herd in cattle farms that had never reared small ruminants indicates that cows can be infected and spread the infection without the presence of the original host. Our results further showed that different genotypes of B. melitensis could be isolated from different samples of the same animal. The local geographic distribution of genotypes showed a very close genetic relatedness with previously reported genotypes outside the study area. Worldwide, all genotypes and strains identified in this study were mostly related to the Western Mediterranean lineage and were less likely to the Americas clonal lineage. In conclusion, uncontrolled animal movement and the ability of B. melitensis to spread among atypical hosts in the absence of the original hosts are potential causes for the failure of brucellosis control programs in endemic areas. The legal importation and illegal movement of cattle and sheep are the main factors for maintaining the infection of B. melitensis within the country. Further investigations are required to understand the reasons for the presence of more than one genotype of B. melitensis in the same animal and the efficacy of the current applied strategy for brucellosis control.

Zoonotic hepatitis E, mainly caused by hepatitis E virus (HEV) genotype (gt) 3, is a foodborne disease that has emerged in Europe in recent decades. The main animal reservoir for genotype 3 is domestic pigs. Pig liver and liver derivates are considered the major risk products, and studies focused on the presence of HEV in pig muscles are scarce. The objective of the present study was to evaluate the presence of HEV in different organs and tissues of 45 apparently healthy pigs from nine Spanish slaughterhouses (50% national production) that could enter into the food supply chain. Anti-HEV antibodies were evaluated in serum by an ELISA test. Ten samples from each animal were analyzed for the presence of HEV RNA by reverse transcription real-time PCR (RT-qPCR). The overall seroprevalence obtained was 73.3% (33/45). From the 450 samples analyzed, a total of 26 RT-qPCR positive samples were identified in the liver (7/45), feces (6/45), kidney (5/45), heart (4/45), serum (3/45), and diaphragm (1/45). This is the first report on detection of HEV RNA in kidney and heart samples of naturally infected pigs. HEV RNA detection was negative for rib, bacon, lean ham, and loin samples. These findings indicate that pig meat could be considered as a low risk material for foodborne HEV infection.

In recent years, cases of hepatitis E virus (HEV) infection have increased in Europe in association with the consumption of contaminated food, mainly from pork products but also from wild boars. The animal's serum is usually tested for the presence of anti-HEV antibodies and viral RNA but, in many cases such as during hunting, an adequate serum sample cannot be obtained. In the present study, liver transudate was evaluated as an alternative matrix to serum for HEV detection. A total of 125 sera and liver transudates were tested by enzyme-linked immunosorbent assay at different dilutions (1:2, 1:10, 1:20), while 58 samples of serum and liver transudate were checked for the presence of HEV RNA by RT-qPCR. Anti- HEV antibodies were detected by ELISA in 68.0% of the serum samples, and in 61.6% of the undiluted transudate, and in 70.4%, 56.8%, and 44.8% of 1:2, 1:10, or 1:20 diluted transudate, respectively. The best results were obtained for the liver transudate at 1:10 dilution, based on the Kappa statistic (0.630) and intraclass correlation coefficient (0.841). HEV RNA was detected by RT-qPCR in 22.4% of the serum samples and 6.9% of the transudate samples, all samples used for RT-qPCR were positive by ELISA. Our results indicate that liver transudate may be an alternative matrix to serum for the detection of anti-HEV antibodies.

El virus de la hepatitis E (VHE) es considerado como el agente principal de hepatitis viral aguda epidemica en paises en vias de desarrollo (se estima que se producen cada ano alrededor de 20 millones de infecciones por el VHE en todo el mundo), mientras que en paises desarrollados se dan casos principalmente esporadicos relacionados, en la mayoria de los casos, con los animales, considerandose actualmente la hepatitis E una zoonosis emergente. Existen cuatro genotipos principales del VHE (de un total de 8) que muestran caracteristicas epidemiologicas y clinicas diferenciadas. El VHE se transmite a menudo a traves del consumo de alimentos tanto de origen animal como vegetal y agua, existiendo factores medioambientales implicados en el ciclo de transmision de este virus. El peligro real de la infeccion por el VHE y de la enfermedad originada dependera no solo del genotipo del virus, si no, tambien, de los diversos escenarios ambientales en las zonas de elevada prevalencia del virus, las condiciones de salubridad e higiene, el tipo de alimento consumido y las costumbres sociales asociadas al consumo de los mismos, ademas de factores profesionales. El desarrollo de estrategias precisas de prevencion y control de la infeccion por VHE debe basarse en avances en el conocimiento de la etiologia, la epidemiologia y los metodos de control del mismo.

La Fiebre Hemorrágica de Crimea-Congo (FHCC) fue descrita por primera vez como entidad clínica en un brote de enfermedad hemorrágica que tuvo lugar entre los años 1944 y 1945 en la Península de Crimea (aunque existen referencias que lo relacionan con episodios del siglo XII), donde alrededor de 200 soldados soviéticos se infectaron mientras asistían a campesinos durante la II Guerra Mundial. Parece que la causa pudo ser la proliferación de liebres en las granjas abandonadas durante la ocupación alemana, coincidiendo con una gran disminución en el número de cabezas de ganado. La explosión de casos entre los soldados y trabajadores de granjas colectivas al reocupar la zona, pueden reflejar la necesidad urgente de los adultos del principal vector del Virus de la Fiebre Hemorrágica del CrimeaCongo (vFHCC), las garrapatas del género Hyalomma, para encontrar un animal de tamaño grande para alimentarse (ver más adelante). Situaciones similares pueden explicar la actual presencia de ”puntos calientes” del vFHCC en otras regiones. Más tarde se comprobó que este proceso estaba causado por un virus, que, posteriormente, se demostró era antigénicamente idéntico a un virus aislado de la sangre de un paciente febril en el Congo Belga (actual República Democrática del Congo) en 1956. Este vínculo otorgó el nombre actual al virus, que pudo finalmente ser cultivado en 1968, tras más de dos décadas de intentos fallidos. Agente cAusAl

Leishmaniasis, caused by Leishmania infantum, is a vector-borne zoonotic disease that is endemic to the Mediterranean basin. The potential of rabbits and hares to serve as competent reservoirs for the disease has recently been demonstrated, although assessment of the importance of their role on disease dynamics is hampered by the absence of quantitative knowledge on the accuracy of diagnostic techniques in these species. A Bayesian latent-class model was used here to estimate the sensitivity and specificity of the Immuno-fluorescence antibody test (IFAT) in serum and a Leishmania-nested PCR (Ln-PCR) in skin for samples collected from 217 rabbits and 70 hares from two different populations in the region of Madrid, Spain. A two-population model, assuming conditional independence between test results and incorporating prior information on the performance of the tests in other animal species obtained from the literature, was used. Two alternative cut-off values were assumed for the interpretation of the IFAT results: 1/50 for conservative and 1/25 for sensitive interpretation. Results suggest that sensitivity and specificity of the IFAT were around 70-80%, whereas the Ln-PCR was highly specific (96%) but had a limited sensitivity (28.9% applying the conservative interpretation and 21.3% with the sensitive one). Prevalence was higher in the rabbit population (50.5% and 72.6%, for the conservative and sensitive interpretation, respectively) than in hares (6.7% and 13.2%). Our results demonstrate that the IFAT may be a useful screening tool for diagnosis of leishmaniasis in rabbits and hares. These results will help to design and implement surveillance programmes in wild species, with the ultimate objective of early detecting and preventing incursions of the disease into domestic and human populations.

Brucellosis is endemic in most parts of Egypt, where it is caused mainly by Brucella melitensis biovar 3, and affects cattle and small ruminants in spite of ongoing efforts devoted to its control. Knowledge of the predominant Brucella species/strains circulating in a region is a prerequisite of a brucellosis control strategy. For this reason a study aiming at the evaluation of the phenotypic and genetic heterogeneity of a panel of 17 Brucella spp. isolates recovered from domestic ruminants (cattle, buffalo, sheep, and goat) from four governorates during a period of five years (2002–2007) was carried out using microbiological tests and molecular biology techniques (PCR, MLVA-15, and sequencing). Thirteen strains were identified as B. melitensis biovar 3 while all phenotypic and genetic techniques classified the remaining isolates as B. abortus (n = 2) and B. suis biovar 1 (n = 2). MLVA-15 yielded a high discriminatory power (h = 0.801), indicating a high genetic diversity among the B. melitensis strains circulating among domestic ruminants in Egypt. This is the first report of the isolation of B. suis from cattle in Egypt which, coupled with the finding of B. abortus, suggests a potential role of livestock as reservoirs of several zoonotic Brucella species in the region.

Antibody detection is the basis of large-scale sheep brucellosis diagnosis because of its sensitivity and specificity. In contrast, information on the cellular mediated immune (CMI) response triggered after Brucella melitensis infection, a cornerstone in the protection against this pathogen, is more limited, particularly regarding the effect of the virulence of the infecting strain in the induced CMI reaction. Here, the interferon-gamma (IFN-γ) profiles evoked after exposure by different routes to virulent (H38) and attenuated (Rev.1) B. melitensis strains in 14 pregnant sheep and 87 ewe lambs, respectively, were characterized accounting for different host-related factors, and compared with their serological response and with the basal IFN-γ responses observed in 155 animals non exposed to Brucella. No significant differences in the IFN-γ response of Rev.1 vaccinated animals depending on the inoculation route was observed, in contrast with their serological results. Response in H38-challenged followed a similar trend although peaked later, and an effect of the abortion on the IFN-γ response was detected. This information could help to understand the interaction bacteria–host that leads to its intracellular survival and could be useful for the design of new diagnostic approaches.

An outbreak of human leishmaniosis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.

DNA-based methods have emerged as an additional tool for Brucella infection-confirmation at a herd level. However, their implementation may require the use of specialized equipment. In this context the recently developed loop-mediated isothermal amplification (LAMP) technique may constitute an additional and cost-effective tool for rapid and specific DNA detection, especially in low income areas. In the present study the usefulness of a newly developed LAMP assay aiming at the multicopy-IS711 sequence was assessed on a variety of clinical samples (n=81 from abortions and ewes; cattle, n=3; swine, n=4) that were analyzed in parallel using real-time PCR and bacteriology. Although overall sensitivities obtained with the three methods were comparable (p>0.05), our results highlighted the complementarity between bacteriology and molecular-based methods for increased sensitivity. Significant differences (p<0.05) were observed with all techniques depending on the nature of the sample. Our results demonstrate the potential of the IS711-LAMP technique for direct Brucella detection.

The problem of estimating the information conveyed about a random variable by another one in a population on the basis of a sample drawn at random and with replacement from it was exhaustively analysed previously. From a theoretical viewpoint, this analysis showed the advantages of the quadratic mutual information against Shannon's mutual information in relation to such a problem. This theoretical conclusion is confirmed from an empirical viewpoint by comparing the population values and estimates for both information measures on the basis of samples obtained from a normal bivariate distribution by a Monte Carlo simulation.