The forest fly (Hippobosca equina) is an obligate haematophagous dipteran insect (order Diptera) that primarily infests horses and may contribute to the circulation of vector-borne pathogens. This study aimed to investigate the presence of Anaplasma phagocytophilum, Borrelia burgdorferi s.l., Babesia caballi, and Theileria equi, important vector-borne pathogens of equids, in forest flies collected from horses in endemic areas of Spain. A total of 170 forest flies were collected from 39 equids across four geographical regions in Spain (Segovia, Madrid, Toledo, and Menorca) and blood samples were collected from 27 of these horses. All flies were morphologically and molecularly identified as H. equina, and DNA extracted from flies and equine blood was screened using multiplex real-time and nested PCR, followed by sequencing and phylogenetic analysis. Neither flies nor horses tested positive for A. phagocytophilum, whereas one fly was positive for B. burgdorferi s.l. (0.6%). In contrast, T. equi and B. caballi DNA were detected in 11.2% and 1.2% of flies, respectively, and all positive flies were collected from horses positive for equine piroplasmosis (T. equi/B. caballi infection), with identical 18S rRNA sequences between hosts and flies. Nested PCR showed a higher detection rate than real-time PCR for the detection of these piroplasms in flies and blood samples. These findings provide the first molecular evidence of EP pathogens in H. equina and support further investigation into the epidemiological importance of forest flies in equine pathogen surveillance.

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.

Background Hippoboscid flies are obligate hematophagous Diptera that infest mammals and birds worldwide. Hippobosca equina, commonly known as the forest fly, primarily affects horses and is considered both a mechanical and/or biological vector of various bacteria, viruses, and protozoa. Equine piroplasmosis (EP) is a tick-borne disease caused by three intra-erythrocytic hemoprotozoans: Theileria equi, Babesia caballi, and the recently identified species Theileria haneyi. To date, no studies have reported the detection of any Piroplasmida in H. equina. This study provides the first evidence of the presence of T. equi and B. caballi in forest flies infesting horses in endemic areas, identified using molecular techniques. Methods A total of 170 forest flies were collected from 35 Equidae across five locations in Spain (Segovia, Madrid, Toledo, and Menorca Island). Blood samples for equine piroplasmosis (EP) testing were drawn from 27 examined horses. All insects were identified both morphologically and molecularly, and genomic DNA from each fly and their equine host blood samples was screened for EP using real-time and nested PCR. Phylogenetic analyses were performed on all obtained T. equi and B. caballi sequences. Results All collected hippoboscids were identified as H. equina using a stereomicroscope and taxonomic keys, as well as by sequencing the mitochondrial cytochrome oxidase subunit 1 (COI) gene. Molecular screening detected T. equi DNA in 19 of 170 (11.2%) collected forest flies and B. caballi DNA in 2 of 170 (1.2%). All EP-positive flies were collected from EP-positive horses, with sequences from the flies and their equine hosts being identical. Two distinct T. equi and two B. caballi sequences were identified. Conclusions This study is the first to report the detection of T. equi and B. caballi in H. equina collected from horses. Given that approximately one-quarter of the flies sampled from EP-positive horses tested positive for the disease, sampling forest flies could be a practical method for detecting equine piroplasmosis in feral horse populations. Experimental transmission studies are required to assess the vector competence of H. equina for EP and other equine diseases.

Equine piroplasmosis (EP) is caused by Theileria equi and Babesia caballi, transmitted by tick vectors. Horses can suffer an acute, subacute, and chronic forms of the disease, with clinical signs such as poor performance, fever, pale mucosal membranes, and jaundice. The diagnosis of EP subclinical cases is complex due to the sensitivity of real-time PCR and the limited parasite load in some carriers, making it challenging to differentiate them from seropositive, PCR negative (S+PCR-) individuals. This study aimed to describe haematological and biochemical changes in asymptomatic EP carriers, EP S+PCR- horses and control horses (EP seronegative and PCR negative). It also investigated potential haemato-biochemical markers to aid in distinguishing true EP carriers alongside molecular and serological tests. A comprehensive haematology and biochemistry profile was conducted on 410 sera and EDTA blood samples, comprising 130 EP positives by real-time PCR and competitive ELISA (cELISA) (carriers), 130 EP negatives by real-time PCR but positive to cELISA (S+PCR-) and 150 EP negative horses to real-time PCR and c-ELISA (controls). Our study confirmed that a haematological and biochemistry profile could help to differentiate between EP carriers/S+PCR- from healthy horses. Carriers and S+PCR- horses showed significant increases in the white blood cell count (WBC), high total proteins (TP) and total globulins (GLOB) concentration, and liver function markers compared to controls. Additionally, the evaluation of uric acid (UA) suggested oxidative stress in carrier horses. However, no useful haemato-biochemical diagnostic markers were identified to aid the challenging differentiation of EP carriers and S+PCR- horses, highlighting the need for improvement in molecular/serological diagnosis for these horses.

Introducción: Cuba adoptó medidas contra la COVID-19 con implicaciones multisectoriales que obligaron a convivir en una nueva normalidad. A principios de 2021 ocurrió un alza de casos en presencia de otras variantes del virus, entre ellas, la Delta del SARS-CoV-2. Objetivo: Valorar la intervención comunitaria diseñada para controlar un brote de COVID-19 en un área de salud de Las Tunas. Material y método: Se realizó una intervención comunitaria implementada en la población que asiste el área de salud del Policlínico Docente Dr Gustavo Aldereguía Lima de Las Tunas compuesta por 53 mil 392 habitantes dispensarizados en el período del 11 de julio de 2021 al 24 de octubre de 2021. Resultados: Se realizaron 7 mil atenciones médicas por IRA, ingresaron en el domicilio 8 mil 314 sospechosos y mil 695 confirmados de COVID-19. Los meses de agosto y octubre fueron los de mayor y menor complejidad por circunscripciones respectivamente. Se notificaron 210,6±136,396 pacientes confirmados por semana con reducción sostenida a partir de la semana estadística 35; posterior a la semana estadística 36 no hubo nuevos fallecidos. Desde la semana estadística 35 se reportaron pacientes inmunizados (26 mil 422) con las vacunas cubanas ABDALA o Soberana Plus. Conclusiones: La intervención comunitaria contribuyó al mejor control del brote por COVID-19 en el área de salud, dado en la disminución de casos y fallecidos por la enfermedad y los niveles de complejidad por circunscripciones.

espanolA finales de febrero y durante todo el mes de marzo, se confirmaron en Espana y varios paises de Europa multiples casos de infeccion por Herpesvirus equino tipo 1 (Equine herpesvirus 1, EHV-1) en caballos que habian participado en competiciones de salto en Espana y Portugal. La infeccion por EHV-1 es endemica en la mayoria de poblaciones de caballos en todo el mundo, y causa perdidas economicas graves asociadas a brotes de enfermedad respiratoria, abortos, muertes neonatales y mieloencefalopatias. EnglishAt the end of February and throughout the month of March, multiple cases of infection with Equine Herpesvirus type 1 (Equine herpesvirus 1, EHV-1) were confirmed in Spain and several European countries in horses that had participated in show jumping competitions in Spain and Portugal. EHV-1 infection is endemic in most horse populations throughout the world, causing severe economic losses associated with outbreaks of respiratory disease, miscarriages, neonatal deaths, and myeloencephalopathies.

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.

La enfermedad COVID-19 es uno de los fenomenos sanitarios mas devastadores de la historia reciente. Dada su reciente descripcion, aun son muchos los aspectos sobre su curso clinico y su agente etiologico (SARS-CoV-2) que se desconocen. Este articulo pretende arrojar luz sobre algunas cuestiones importantes acerca de su posible origen y permanencia, asi como sobre la produccion de vacunas frente a su agente causal a traves de un estudio comparativo basado en la informacion disponible sobre coronavirus animales, bien conocidos por los veterinarios desde hace casi un siglo.

espanolDurante los ultimos meses hemos vivido una situacion sanitaria excepcional en la que la aparicion de un nuevo patogeno altamente transmisible ha colapsado los sistemas de salud y de vigilancia de muchos paises. En las primeras fases de la pandemia de COVID-19 resulto evidente que una gestion eficaz de esta crisis requeria informacion sobre el estado infectivo de la poblacion. Ante la evidente saturacion de la capacidad diagnostica de nuestro pais, en la Facultad de Veterinaria de la UCM nace una iniciativa de apoyo al sistema sanitario que cristaliza en forma de red de laboratorios dedicados a la realizacion de tests PCR para deteccion del virus SARS-CoV-2. Su actividad se centra en el grupo mas vulnerable de nuestra region: los ancianos de las residencias de mayores de la Comunidad de Madrid. Tras tres meses de trabajo, la informacion aportada por la red ha contribuido a la correcta gestion epidemiologica de estos centros, ayudando a los sanitarios a prevenir nuevas infecciones y, en definitiva, a salvar vidas. Esta es la historia de como un grupo de investigadores de diferentes ramas se unen de forma desinteresada para apoyar a un sistema sanitario sometido a un nivel de exigencia inesperado y extremo con un unico objetivo en mente: ayudar. EnglishIn recent months we have experienced an exceptional health situation in which the appearance of a new highly communicable pathogen has collapsed the health and surveillance systems of many countries. In the early phases of the COVID-19 pandemic, it became clear that effective management of this crisis required information on the infective status of the population. Faced with the evident saturation of the diagnostic capacity of our country, an initiative to support the health system was born at the UCM Faculty of Veterinary Medicine, which crystallizes in the form of a network of laboratories dedicated to carrying out PCR tests for detection of the SARS-CoV virus. -2. Its activity is focused on the most vulnerable group in our region: the elderly in nursing homes in the Community of Madrid. After three months of work, the information provided by the network has contributed to the correct epidemiological management of these centers, helping health workers to prevent new infections and, ultimately, to save lives. This is the story of how a group of researchers from different branches they unselfishly come together to support a healthcare system subjected to an unexpected and extreme level of demand with a single goal in mind: to help.

Borna disease virus (BoDV) can cause neurological disease in several vertebrate species, especially in horses and sheep.The recent cases of fatal encephalitis caused by BoDV in humans have raised concern regarding the zoonotic character of this virus.BoDV infection in horses is endemic in Central Europe, whereas in Spain the disease is regarded as an exotic disease in horses and serological investigations in these animals have never been performed.The aim of this study was to investigate the exposure to BoDV in breeding and sport horses in Spain and to determine risk factors for the transmission of the virus.Serum samples from 495 horses (260 sport horses and 235 Spanish Purebred breeding horses) were tested by an Indirect Fluorescence Antibody Test (IFAT).From the SP breeding horses we examined factors putatively associated with seropositivity to BoDV.A total of 164 horses (33.1%; 95% CI 29.0-37.3)showed antibodies against BoDV.Factors associated with BoDV seropositivity in the SP breeding horses were climate, age and the time of the year when the horses were sampled.This is the first report on the presence of BoDV antibodies in apparently asymptomatic breeding and sport horses in Spain.Our results demonstrate the need for BoDV surveillance in horses and possible reservoir hosts in this country, and suggests that BoDV should be included in the differential diagnosis of equine and human cases of encephalitis.

Serological analysis of equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is included in the export testing requirements for most of the countries worldwide, thus involving a high economic impact on equine industry of EP-endemic countries, such as Spain. A total of 3368 serum samples from healthy horses collected prior to export between 2015 and 2018 in Spain were tested for antibodies against T. equi and B. caballi by using a competitive inhibition enzyme-linked immunosorbent assay (cELISA). The overall seroprevalence results in Spain revealed that almost a quarter of the horses analysed (24.1 %; 95% CI 22.6-25.5) could not be exported to countries free from EP. The implementation of prevention measures such as the use of acaricides and daily checks for ticks in horses, as well as regular serological screening of horses in Spain would aid to increase the number of horses exported to other countries.

The application of MALDI-TOF MS for identifying streptococcal isolates recovered from clinical specimens of diseased pigs was evaluated. For this proposal, the MALDI BDAL Database (Bruker Daltoniks, Germany) was supplemented with the main spectrum profiles (MSP) of the reference strains of S. porci, S. porcorum and S. plurextorum associated with pneumonia and septicemia. Although these three species showed similar MALDI profiles, several peaks were recognized that can be useful for their differentiation: S. porci (4113, 6133, 7975 and 8228 m/z Da), S. plurextorum (3979, 4078, 4665, 6164, 6491, 6812, 7959 and 9330 m/z Da) and S. porcorum (3385, 3954, 4190, 6772, 7908, and 8381 m/z Da). After adding these MSPs, an evaluation was conducted to determine the accuracy of MALDI-TOF MS for the identification of streptococci from diseased pigs using 74 field isolates. Isolates were identified as S. suis, S. porcinus, S. dysgalactiae, S. hyovaginalis, S. porcorum, S. alactolyticus, S. hyointestinalis and S. orisratti. This is the first time that the latter three species have been reported from clinical specimens of pigs. Overall, there was good concordance (95.9%) between the results obtained from MALDI-TOF MS identification (best hint) and those from genotyping. Our results demonstrate the good performance of MALDI-TOF MS (100% sensitivity and specificity) for identifying most of the species of streptococci that can frequently be isolated from diseased pigs. However, conflicting results were observed in the correct identification of some isolates of S. dysgalactiae and S. alactolyticus.

The aim of this study was to compare a quantitative real-time PCR (qPCR) validated for the detection of Leishmania infantum in dogs with a nested PCR but in wild Leporidae. Additionally, L. infantum results from indirect immunofluorescent antibody test (IFAT) and in vitro culture were also compared with qPCR. Different samples (spleen, skin and hair) recovered from 224 European rabbits and 70 Iberian hares from two green areas of Madrid Council were analysed for the detection of L. infantum. The presence of Leishmania kDNA was detected by qPCR in 58 out of 221 (26.24%), 162 out of 203 (79.8%) and 22 out of 33 (66.67%) analysed rabbits on spleen, skin and hair samples, respectively; and in 7 out of 69 (10.14%), 39 out of 70 (55.71%) and 17 out of 32 (53.13%) test hares on spleen, skin and hair samples, respectively. The qPCR in all test samples resulted to be more sensitive than nested PCR, with a limit of detection of 1.43 fg/reaction (0.039 parasites) for L. infantum genomic DNA. Additionally, the percentage of positive animals detected by qPCR in at least two out of three samples (n=221 rabbits and 70 hares) tested was higher than those detected by IFAT (n=190 rabbits and 61 hares) and isolation (n=75 rabbits and 20 hares). The highest level of agreement was obtained by nested PCR on spleen/skin (89%/83%) samples and qPCR on spleen samples (81%), followed by IFAT (48%) and qPCR on skin (32%) samples. Our results demonstrate this qPCR is a suitable method for detecting L. infantum DNA in different samples suggesting hair could be considered an adequate sample for direct, reliable and non-invasive diagnosis of L. infantum in wild animals.

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.

RB51 vaccination can minimise the diagnostic problems associated with S19 vaccination of adult cattle, but its use for bovine brucellosis (BB) control remains controversial. Here, the evolution of BB prevalence in five high prevalence areas in Spain subjected to different control measures is described: herd depopulation of infected herds (I-III) or mass vaccination with RB51 and S19-RB51 vaccination of replacement heifers (IV-V). Annual data from the eradication campaigns were analysed at the special incidence area (SIA) level and the time to obtain herd prevalence levels of <1 per cent ('controlled status') was obtained at the local veterinary unit (LVU) level and compared using Cox's proportional hazard model. A higher annual rate of decrease in herd prevalence was observed in the SIAs subjected to vaccination (46.9%, 95% CI 43.5% to 50.0%) compared with those managed using stamping out (14.9%, 95% CI 9.6% to 19.9%). No significant differences in the time to achieve controlled status were observed between the stamping-out and vaccination strategies used at the LVU level, with median times of 60 (stamping-out LVUs) and 63 (vaccination LVUs) months. These results suggest that RB51 mass vaccination, in combination with the S19-RB51 vaccination of replacement heifers and strict implementation of other eradication measures, may provide results at least comparable with those resulting from a herd depopulation based strategy.

Brucella melitensis is a major human and animal pathogen, with a wide host range that includes all domestic ruminant species, although small ruminants are its preferred hosts. Outbreaks in cattle due to B. melitensis have become a worldwide emerging problem particularly difficult to control due to the lack of knowledge on the epidemiology in this host species and of an effective vaccine. However, combination of molecular tools and strict biosecurity measures can help to solve these difficulties and eventually eradicate the disease from infected herds. In the present report, management of an outbreak in Spain involving four farms, more than 2000 cattle and several human cases is described. Application of Multiple Locus VNTR Analysis (MLVA) allowed identifying the most likely source of infection. Stamping out and test-and-slaughter strategies were applied, proving their usefulness to control the outbreak depending on infection level, and without the need of other alternative measures.

Spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis are the international standard techniques for molecular typing of members of the Mycobacterium tuberculosis complex. To enable the exploitation of molecular typing data for epidemiological purposes, the creation of large databases is indispensable. Here we describe mycoDB.es, a database for animal tuberculosis which forms part of the Spanish national programme for eradication of bovine tuberculosis. This database has been created as an epidemiological tool at national level and contains spoligotype patterns of 17,273 isolates clustered in 401 different spoligotypes of Mycobacterium bovis, Mycobacterium caprae and M. tuberculosis. The database offers an overview of the present spoligotypes, to a lower extent also of MIRU-VNTR types, affected animal species and furthermore of the spatial distribution of these genotypes.