1. Investigación

Listeria monocytogenes is a major human and animal foodborne patho-gen. However, data from environmental reservoirs remain scarce. Here, we usedwhole-genome sequencing to characterize Listeria species isolates recovered over 1year from wild animals in their natural habitats in Spain. Three different Listeria spp.(L. monocytogenes [n = 19], Listeria ivanovii subsp. londoniensis [n = 4], and Listeriainnocua [n = 3]) were detected in 23 animal tonsils (9 deer, 14 wild boars) and 2feeding troughs. No Listeria species was detected in feces. L. monocytogenes wasdetected in tonsils of 44.4% (8 out of 18) of deer and 40.7% (11 out of 27) of wildboars. L. monocytogenes isolates belonged to 3 different core genome multilocussequence typing (cgMLST) types (CTs) of 3 distinct sublineages (SL1, SL387, andSL155) from lineages I and II. While cgMLST type L1-SL1-ST1-CT5279 (IVb; clonalcomplex 1 [CC1]) occurred only in one animal, types L1-SL387-ST388-CT5239 (IVb;CC388) and L2-SL155-ST155-CT1170 (IIa; CC155) were retrieved from multiple ani-mals. In addition, L1-SL387-ST388-CT5239 (IVb; CC388) isolates were collected 1 yearapart, revealing their long-term occurrence within the animal population and/orenvironmental reservoir. The presence of identical L. monocytogenes strains in deerand wild boars suggests contamination from a common food or environmentalsource, although interhost transmission cannot be excluded. Pathogenicity islandsLIPI-1, LIPI-3, and LIPI-4 were present in 100%, 5%, and 79% of the L. monocytogenesisolates, respectively, and all L. monocytogenes lineage II isolates (n = 3) carried SSI-1stress islands. This study highlights the need for monitoring L. monocytogenes envi-ronmental contamination and the importance of tonsils as a possible L. monocyto-genes intrahost reservoir.

Background: Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis, an infectious disease in animals and people, with pigs acting as asymptomatic reservoirs. In August 2019 an outbreak associated with the consumption of pork meat caused 222 human cases of listeriosis in Spain. Determining the diversity as well as the virulence potential of strains from pigs is important to public health. Methods: The behaviour of 23 L monocytogenes strains recovered from pig tonsils, meat and skin was compared by studying (1) internalin A, internalin B, listeriolysin O, actin assembly-inducing protein and PrfA expression levels, and (2) their invasion and intracellular growth in eukaryotic cells. Results: Marked differences were found in the expression of the selected virulence factors and the invasion and intracellular replication phenotypes of L monocytogenes strains. Strains obtained from meat samples and belonging to serotype 1/2a did not have internalin A anchored to the peptidoglycan. Some strains expressed higher levels of the studied virulence factors and invaded and replicated intracellularly more efficiently than an epidemic L monocytogenes reference strain (F2365). Conclusion: This study demonstrates the presence of virulent L monocytogenes strains with virulent potential in pigs, with valuable implications in veterinary medicine and food safety.

Listeriosis is a zoonotic disease caused by Listeria monocytogenes and Listeria ivanovii. The genus Listeria currently includes 27 recognized species and is found throughout the environment. The number of systematic studies on antimicrobial resistance in L. monocytogenes isolates from domestic farms using antimicrobial substances is limited. Importantly, dairy ruminant farms are reservoir of hypervirulent lineage I L. monocytogenes isolates, previously associated with human clinical cases. Considering that the classes of antibiotics used in food-producing domestic animals are frequently the same or closely related to those used in human medicine, studies about the impact of antibiotic use on the acquisition of antibiotic resistance in Listeria spp. in domestic animal farms are, therefore, of high importance. Here, susceptibility to 25 antibiotics was determined. Eighty-one animal-related, 35 food and 21 human pathogenic Listeria spp. isolates and 114 animal-related non-pathogenic Listeria spp. isolates were tested. Whole genome sequencing data was used for molecular characterization. Regarding L. monocytogenes, 2 strains from the clinical-associated linage I showed resistance to erythromycin, both related to dairy ruminants. Acquired resistance to one antibiotic was exhibited in 1.5% of L. monocytogenes isolates compared with 14% of non-pathogenic Listeria spp. isolates. Resistance to tetracycline (7.9%), doxycycline (7.9%), penicillin (4.4%), and ampicillin (4.4%) were the most frequently observed in non-pathogenic Listeria spp. While resistance to two or more antibiotics (5.6%) was most common in Listeria spp., isolates, resistance to one antibiotic was also observed (1.6%). The present results show that non-pathogenic Listeria spp. harbour antimicrobial resistance genes.

Toxoplasmosis is one of the most common parasitic zoonoses and represents a significant health risk for humans, especially for immunodeficient patients. The main transmission route is by oral uptake of oocysts and consumption of undercooked meat of infected animals. Different species have been evaluated as possible reservoirs of the parasite, but few studies have been carried out to examine the role of horses in transmission of the disease. Given the proximity of these animals to humans and the widespread consumption of their meat in many countries, including the Mediterranean basin, it is important to determine the prevalence of T. gondii infection in this species. In this study, blood samples from 105 horses were collected and the presence of T. gondii was evaluated by serological and molecular methods. Antibodies against T. gondii of 12 horses (11.43%) were detected by enzyme-linked immunosorbent assay (ELISA), whereas 29 horses (27.62%) showed positive for PCR. Seroprevalence was related to use of the animals, being higher in horses used for dressage than in others. Purebreds had higher seroprevalence than crossbred animals. No differences between breed, sex or age were found. The results of this study confirm the presence of T. gondii infection in horses, highlighting the need to analyse the meat of this species before human consumption and to control of this infection in horses, as they could be an important reservoir of this zoonotic parasite.

Leishmania is a genus of parasitic protozoa that causes a disease called leishmaniasis. Leishmaniasis is transmitted to humans through the bites of infected female sandflies. There are several different species of Leishmania that can cause various forms of the disease, and the symptoms can range from mild to severe, depending on species of Leishmania involved and the immune response of the host. Leishmania parasites have a variety of reservoirs, including humans, domestic animals, horses, rodents, wild animals, birds, and reptiles. Leishmaniasis is endemic of 90 countries, mainly in South American, East and West Africa, Mediterranean region, Indian subcontinent, and Central Asia. In recent years, cases have been detected in other countries, and it is already an infection present throughout the world. The increase in temperatures due to climate change makes it possible for sandflies to appear in countries with traditionally colder regions, and the easy movement of people and animals today, facilitate the appearance of Leishmania species in new countries. These data mean that leishmaniasis will probably become an emerging zoonosis and a public health problem in the coming years, which we must consider controlling it from a One Health point of view. This review summarizes the prevalence of Leishmania spp. around the world and the current knowledge regarding the animals that could be reservoirs of the parasite.

Leishmaniasis is a zoonotic parasitic disease, and the main reservoir of the parasite is the dog, although recent years have seen an increase in other mammalian species. In the Mediterranean region, where it is an endemic disease, it is caused by the species Leishmania infantum. The Ibizan hound, an autochthonous breed of this region, appears to have a genetic resistance to parasitic infection, whereas other canine breeds, such as the Boxer, are susceptible to infection. These differences are related to the differentiated activation of the immune response, with the Ibizan hound activating the Th1 immune response, whereas the Boxer breed triggers the Th2 immune response. Cytokine levels and genomic haplotypes of several genes involved in the immune response were analysed in twenty-eight Ibizan hound (resistant canine breed model) and twenty-four Boxer (susceptible canine breed) without clinical signs in the Mediterranean region. Cytokine levels were analysed by ELISA commercial kits and haplotypes were studied using CanineHD DNA Analysis BeadChip including 165,480 mapped positions. The results show 126 haplotypes associated with differential immune response in dogs. Specifically, haplotypes in IL12RB1, IL6R, CIITA, THEMIS, NOXA1, HEY2, RAB38, SLC35D2, SLC28A3, RASEF and DAPK1 genes are associated with serum levels of IFN-γ, IL-2, IL-8, and IL-18. These results suggest that the resistance or susceptibility to Leishmania infantum infection could be a consequence of haplotypes in several genes related to immune response. Future studies are needed to elucidate the relationship of these haplotypes with immune response and gene expression regulation.

Leishmaniosis is a zoonosis caused by Leishmania spp., an intracellular protozoan parasite. This parasite is transmitted by sandflies, and the disease is endemic in the Mediterranean basin. In recent years, the number of species which could be a reservoir of the parasite is increased. One of the most relevant species is the horse, due to their contact with humans and ability to control the disease, thus being a possible silent reservoir. In this study, we have analyzed the prevalence and factors related to L. infantum infection in healthy horses in the Mediterranean region. Epidemiological data and serum samples were obtained from 167 apparently healthy horses, and the presence of L. infantum was evaluated via the ELISA method and real-time PCR. The results show 27.5% of prevalence and that the main factors related to infection are equine breed, morphotype, outdoor living, use, and season. In conclusion, the prevalence of L. infantum infection in apparently healthy horses from eastern Spain (Mediterranean basin) is elevated. To control this zoonosis, it would be advisable to carry out more studies on this and other species that could be silent reservoirs of the parasite, as well as carry out measures such as the use of repellents on a regular basis.

El pescado tiene una relevancia fundamental en la dieta de los seres humanos, sin embargo, su consumo no está exento de riesgos, entre ellos, la presencia de parásitos responsables de enfermedades zoonósicas. Los peces marinos pueden albergar parásitos unicelulares emergentes, de origen terrestre y transmisión hídrica (EWUP, siglas en inglés), sin estar necesariamente infectados. Por otro lado, los peces marinos son verdaderos hospedadores de parásitos de reconocida importancia en salud pública, destacando el nematodo Anisakis pegreffii en el Mediterráneo, causante de la anisakiosis. La primera parte de esta tesis doctoral se enfoca en la detección y caracterización molecular (PCR y metabarcoding) de algunos EWUP de importancia en peces de consumo (cultivados y silvestres), de la costa española del Mediterráneo. Se detectaron especies y subtipos zoonósicos de Cryptosporidium, Blastocystis y microsporidios, aunque en prevalencias muy bajas, sugiriendo un limitado riesgo de transmisión por consumo de pescado procedente de las poblaciones estudiadas. En la segunda parte, se propone un protocolo in vitro para el desarrollo larvario temprano de A. pegreffii, complementado con un estudio ultraestructural. Esta herramienta, como parte del ciclo biológico in vitro de A. pegreffii, puede ser muy útil para la obtención de material biológico destinado a futuros estudios de patogenicidad, diagnóstico o terapéutica.

Free-living cats usually live in colonies in urban areas, especially close to parks and neighbourhoods where people feed them without any sanitary control. This can pose a human, animal and environmental health concern due to the close contact between uncontrolled colonies, the population and other domestic and/or wild animals. Thus, this study aimed to assess the genetic diversity and antimicrobial resistance (AMR) among Salmonella enterica subsp. enterica strains isolated from feral cats in a previous epidemiological study in the Gran Canaria island (Spain). A total of nineteen Salmonella isolates were obtained from November 2018 to January 2019 in a Salmonella epidemiological study in feral cats. All isolates obtained were genotyped by pulsed-field gel electrophoresis (PGFE) and were tested for antimicrobial susceptibility, in accordance with Decision 2013/652/EU. PFGE analysis revealed isolates clustering by serovar, with identical clones for serovars Bredeney and Grancanaria, while differing pulsotypes were observed for serovars Florida (88.89 % similarity) and Nima (83.23 % similarity). All but two isolates were resistant to at least one antimicrobial. The results obtained demonstrate that feral cats in the region investigated are a reservoir of Salmonella strains resistant to gentamicin (94.1 %) and of the critically important antimicrobial tigecycline (23.5 %). Hence, they could excrete AMR strains through their faeces and contaminate the environment, favoring the spread of such bacteria to cohabiting pets. Moreover, this widespread presence of AMR Salmonella clones across various serovars highlights the urgent need to implement efficient antimicrobial stewardship and control programs by the local governments due to the ongoing need to protect human and animal health under a One Health concept.

Dogs are considered the main reservoir of several zoonoses endemic to the Mediterranean Basin. In this study, a prevalence of infections and coinfections of canine vector-borne diseases was analyzed in apparently healthy dogs of different canine pure breeds in Sicily (Italy), where these diseases are endemic. The seroprevalence of Leishmania infantum, Ricketsia ricketsii, Anaplasma phagocytophilum, and Erlichia canis was assessed, as single and coinfections. Biochemical and hematological parameters were evaluated, and epidemiological factors, including sex, age, and canine breed, were recovered. The most frequent infection was L. infantum (45.61%), following R. ricketsii (36.84%), both as single, double, or triple coinfections. Coinfections change the biochemical and hematological parameters of the host, and canine breeds are related to the infection frequency and the parameters observed during infections. Changes in the complete blood count (CBC) and biochemical values also differ between canine breeds, with the Cirneco dell’Etna dogs infected with L. infantum being the animals presenting the most interesting results in our study. High values of RBC, hemoglobin, hematocrit, mean corpuscular hemoglobin (MCH), the albumin/globulin (A/G) ratio, and albumin and low levels of β-2 globulin and γ-globulin were found only in this canine breed, suggesting some resistance to infection in these dogs. Future studies about the immune response of this canine breed could be interesting to determine their possible resistance to zoonotic pathogens, such as L. infantum.