Person: Martínez Alares, Irene
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First Name
Irene
Last Name
Martínez Alares
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Veterinaria
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Item Detection of Brucella in Dermacentor Ticks of Wild Boar with Brucellosis(Transboundary and Emerging Diseases, 2024) Rebollada Merino, Agustín Miguel; Martínez Alares, Irene; Duque, Clara; García-Seco Romero, María Teresa; Escacena, Cristina; Domínguez Rodríguez, Lucas José; Rodríguez Bertos, Antonio Manuel; García Benzaquén, Nerea; Daniel DiazBrucellosis 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.Item Development and evaluation of an IS711-based loop mediated isothermal amplification method (LAMP) for detection of Brucella spp. on clinical samples(Research in Veterinary Science, 2013) Pérez Sancho, Marta; García-Seco Romero, María Teresa; Arrogante, L; García, N; García Benzaquén, Nerea; Martínez Alares, Irene; Díez Guerrier, Alberto Antoine; Perales, A; Goyache Goñi, Joaquín; Domínguez Rodríguez, Lucas José; Álvarez Sánchez, JulioDNA-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.Item Interferon-gamma responses in sheep exposed to virulent and attenuated Brucella melitensis strains(Veterinary Immunology and Immunopathology, 2014) Pérez Sancho, Marta; Durán-Ferrer, Manuel; García-Seco Romero, María Teresa; Macías, Paula; García Benzaquén, Nerea; Martínez Alares, Irene; Ruiz, Elena; Legaz, Emilio; Díez Guerrier, Alberto Antoine; González Domínguez, Sergio; Domínguez Rodríguez, Lucas José; Álvarez Sánchez, JulioAntibody 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-gamma) 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-gamma responses observed in 155 animals non exposed to Brucella. No significant differences in the IFN-gamma 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-gamma 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.Item Liver Transudate, a Potential Alternative to Detect Anti-Hepatitis E Virus Antibodies in Pigs and Wild Boars (Sus scrofa)(Microorganisms, 2020) Navarro Gómez, Alejandro; Bárcena Asensio, María Carmen; Pozo Piñol, Pilar; Díez Guerrier, Alberto Antoine; Martínez Alares, Irene; Polo, Coral; Duque, Clara; Rodríguez-Lázaro, David; Goyache Goñi, Joaquín; García Benzaquén, NereaIn 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.Item Assessment of genetic diversity of zoonotic Brucella spp. recovered from livestock in Egypt using multiple locus VNTR analysis(BioMed research international, 2014) Menshawy, Ahmed M S; Hosein, Hosein I; García Benzaquén, Nerea; Martínez Alares, Irene; Sayour, Ashraf E; Goyache Goñi, Joaquín; Azzam, Ragab A A; Álvarez Sánchez, Julio; Domínguez Rodríguez, Lucas José; Pérez Sancho, Marta; García-Seco Romero, María Teresa; Pérez Sancho, MartaBrucellosis 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.Item Environment and Offspring Surveillance in Porcine Brucellosis(Frontiers in Veterinary Science, 2022) Rebollada Merino, Agustín Miguel; Pérez Sancho, Marta; Rodríguez Bertos, Antonio Manuel; García Benzaquén, Nerea; Martínez Alares, Irene; Navarro Gómez, Alejandro; Domínguez Rodríguez, Lucas José; García-Seco Romero, María TeresaPorcine brucellosis, caused by Brucella suis (B. suis), is a notifiable disease causing significant economic losses in production systems.Most infected pigsmay 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.Item Occurrence of Hepatitis E Virus in Pigs and Pork Cuts and Organs at the Time of Slaughter, Spain, 2017(Frontiers in Veterinary Science, 2020) García Benzaquén, Nerea; Marta Hernández; Maialen Gutierrez-Boada; Antonio Valero; Navarro Gómez, Alejandro; Milagros Muñoz-Chimeno; Fernández Manzano, Álvaro; Franco Matías Escobar; Martínez Alares, Irene; Bárcena Asensio, María Carmen; González Domínguez, Sergio; Ana Avellón; Jose M. Eiros; Gislaine Fongaro; Domínguez Rodríguez, Lucas José; Goyache Goñi, Joaquín; David Rodríguez-LázaroZoonotic 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 realtime 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.Item Trans‐species transmission of Brucellae among ruminants hampering brucellosis control efforts in Egypt(Journal of Applied Microbiology, 2022) Yamen Mohammed Hegazy; Nour Hosny Abdel‐Hamid; Mohammed Eldehiey; Atef F. Oreiby; Magdy Hasanian Algabbary; Mahmoud E. R. Hamdy; Eman Ibrahim Beleta; Martínez Alares, Irene; Momtaz Abdelhady Shahein; García Benzaquén, Nerea; Mahmoud EltholthAims: This study aimed to identify the genotypic fingerprinting of Brucella melitensis biovar 3 isolates from ruminants in Kafr El-Sheikh, Egypt, to compare with other peers globally and to highlight the epidemiology and potential causes of brucellosis control failure.Item Assessment of the sensitivity and specificity of serological (IFAT) and molecular (direct PCR) techniques for diagnosis of leishmaniasis in lagomorphs using a Bayesian approach(Veterinary Medicine and Science, 2016) Cruz Conty, María Luisa de la; Pérez, Andres; Domínguez, Mercedes; Moreno, Inmaculada; García Benzaquén, Nerea; Martínez Alares, Irene; Navarro Gómez, Alejandro; Álvarez, Julio; Domínguez Rodríguez, Lucas JoséLeishmaniasis, 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.