Occurrence of Anaplasma spp. in wild lagomorphs from Southern Spain: Molecular detection of new Anaplasma bovis lineages.

Wild lagomorphs can act as reservoirs of several pathogens of public and animal health concern. However, the number of studies assessing the presence of Anaplasma spp. in these species is scarce. The aim of the present study was to molecularly identify Anaplasma spp. in wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis) from Southern Spain and assess their epidemiological role in the maintenance of the bacterium. During 2017-2021, spleen samples of 394 wild rabbits and 145 Iberian hares were collected. Anaplasma DNA was detected using different PCR assays (16S rRNA and groEL) and phylogenetic analyses were carried out by Bayesian approach. The possible influence of lagomorph species, age and sex on the prevalence of Anaplasma spp. was evaluated by a multiple logistic regression model. The 9.4% of the rabbits were positive to Anaplasma bovis, but all the hares were negative. No significant differences were found in Anaplasma spp. prevalence regarding to age or sex. This is the first report of A. bovis in lagomorphs from Europe. The phylogenetic analysis of A. bovis confirms the existence of different clusters suggesting the existence of several lineages. In addition, a high divergence of nucleotide identity was observed within the lineage 4, which could result in the under-detection of some strains when using A. bovis-specific PCR, hindering its detection and characterization. Since this analysis is based on a limited number of nucleotide bases and sequences, more studies are needed for further characterize A. bovis, as well as its relationship with other Anaplasma spp.

Molecular detection of Rickettsia spp. in wild ungulates and their ticks in Mediterranean areas of southwestern Spain.

Wildlife is an important reservoir of zoonotic pathogens. The objective of the present study was to assess the importance of wild ungulates in the epidemiology of Rickettsia spp. Ticks and spleen samples were collected from 262 red deer (Cervus elaphus) and 83 wild boar (Sus scrofa) hunted in southwestern Spain over a 5-year period. DNA was extracted from tick pools (n = 191) and spleens (n = 345), and two nested PCR assays targeting the rOmpA and rOmpB genes were used to detect Rickettsia DNA. Five tick species were identified (Hyalomma lusitanicum, Dermacentor marginatus, Ixodes ricinus, Rhipicephalus bursa and Haemaphysalis sulcata). Rickettsia DNA was detected in 31 (16.2%) tick pools and two red deer spleen samples (0.8%). Four validated Rickettsia species (R. slovaca, R. monacensis, R. helvetica and R. raoultii), one uncultivated species (Candidatus R. rioja) and two uncharacterized Rickettsia spp. were detected in ticks. R. helvetica and R. slovaca were also detected in spleen samples from red deer. The overall prevalence in ungulate spleen samples was lower than in tick pools suggesting that these ungulates do not play a major role in the transmission of Rickettsia spp. However, their importance as spreaders of positive ticks cannot be ruled out. The results present a challenge for the veterinary and public health communities since most of the Rickettsia spp. detected are pathogenic. Furthermore, the new Rickettsia species could be potential pathogens. For these reasons, identifying Rickettsia species present in ticks and wildlife is of particular interest to clarify their sylvatic cycle and establish appropriate control measures.

Occurrence and limited zoonotic potential of Cryptosporidium spp., Giardia duodenalis, and Balantioides coli infections in free-ranging and farmed wild ungulates in Spain.

Little information is currently available on the occurrence and molecular diversity of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Balantioides coli in wild ungulates and the role of these host species as potential sources of environmental contamination and consequent human infections. The presence of these three pathogens was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were retrospectively collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from the five Spanish bioregions. Overall infection rates were 3.0% (42/1382; 95% CI: 2.1-3.9%) for Cryptosporidium spp., 5.4% (74/1382; 95% CI: 4.2-6.5%) for G. duodenalis, and 0.7% (9/1382; 95% CI: 0.3-1.2%) for B. coli. Cryptosporidium infection was detected in roe deer (7.5%), wild boar (7.0%) and red deer (1.5%), and G. duodenalis in southern chamois (12.9%), mouflon (10.0%), Iberian wild goat (9.0%), roe deer (7.5%), wild boar (5.6%), fallow deer (5.2%) and red deer (3.8%). Balantioides coli was only detected in wild boar (2.5%, 9/359). Sequence analyses revealed the presence of six distinct Cryptosporidium species: C. ryanae in red deer, roe deer, and wild boar; C. parvum in red deer and wild boar; C. ubiquitum in roe deer; C. scrofarum in wild boar; C. canis in roe deer; and C. suis in red deer. Zoonotic assemblages A and B were detected in wild boar and red deer, respectively. Ungulate-adapted assemblage E was identified in mouflon, red deer, and southern chamois. Attempts to genotype samples positive for B. coli failed. Sporadic infections by canine- or swine-adapted species may be indicative of potential cross-species transmission, although spurious infections cannot be ruled out. Molecular evidence gathered is consistent with parasite mild infections and limited environmental contamination with (oo)cysts. Free-ranging wild ungulate species would not presumably play a significant role as source of human infections by these pathogens. Wild ruminants do not seem to be susceptible hosts for B. coli.

Pedicle myiasis by Lucilia caesar (Diptera, Calliphoridae): An emerging disease in roe deer from north-western Spain.

A total of 35 male roe deer (Capreolus capreolus) from Lugo province (north-western Spain) were examined for pedicle myiasis between 2020 and 2022. All these animals had died by different causes and were examined or preserved just after their death to avoid post-mortem infestations. After external and internal head inspection, five animals were diagnosed with severe myiasis at the basis of the antlers (14.29%; 95% confidence interval = 5.38-31.04). All the affected bucks presented a cutaneous wound (1.5-15 cm in diameter) around the pedicles with extensive tissue destruction, exposition of frontal and parietal bones, and massive infestation by dipteran larvae (2-12 mm long). Four of five roe deer showed whitish egg clusters adhered to the basis of the antlers. Moreover, in one animal, six larvae had penetrated the cranial cavity, and in two of them larvae were also found in the nasopharyngeal cavity. All the cases were recorded in summer (May-August). Morphological identification and subsequent molecular confirmation revealed that all animals were infested by different larval stages of Lucilia caesar (Diptera: Calliphoridae). This study represents the first report of pedicle myiasis in Spain. Since this disease is reported sporadically, the detection of five cases in a short period of time suggests an increase in the incidence of this myiasis.

Molecular detection of Ehrlichia spp. in ticks parasitizing wild lagomorphs from Spain: characterization of a novel Ehrlichia species.

BACKGROUND: Several species belonging to the genus Ehrlichia are considered pathogenic for animals and humans. Although wildlife are known to play an important role in the epidemiology of these bacteria, information on the role of wild lagomorphs in their sylvatic cycle is limited. Thus, the objective of the present study was to assess the occurrence of Ehrlichia spp. in ticks collected from wild lagomorphs in Spanish Mediterranean ecosystems. METHODS: A total of 1122 pooled ticks (254 pools) collected from 506 wild rabbits (Oryctolagus cuniculus) and 29 Iberian hares (Lepus granatensis) were analysed using a nested PCR assay targeting the partial groEL gene. Ehrlichia spp.-positive samples were further subjected to a second PCR assay targeting 16S rRNA. RESULTS: Three (1.2%) tick pools comprising Rhipicephalus pusillus collected from nine wild rabbits were positive for Ehrlichia spp. All the Ehrlichia DNA sequences were identical, and use of sequence and phylogenetic analyses allowed us to identify a novel Ehrlichia species. CONCLUSIONS: We provide evidence that a novel Ehrlichia species, named herein as 'Candidatus Ehrlichia andalusi', which may be of concern for animal and public health, is circulating in R. pusillus in Spanish Mediterranean ecosystems. Further studies are warranted to assess the epidemiology, pathogenicity and zoonotic potential of this Ehrlichia species.

Presence of Porcine Circovirus Type 2 in the Environment of Farm Facilities without Pigs in Long Term-Vaccinated Farrow-to-Wean Farms.

Vaccination against Porcine Circovirus Type 2 (PCV2) even over several years has proven as an insufficient measure to eradicate the infection from farms, possibly due to not producing sterilizing immunity. Viral persistence in the farm environment has been proposed as a possible cause of reinfection, and for that reason, the main objective of this study was to identify potential critical points where PCV2 could persist in farrow-to-wean farms which had been vaccinating piglets for years. Surface samples were collected from different farm facilities with and without animals and analyzed by qPCR to detect and quantify the viral load. Most of the samples taken in animal housing facilities tested negative (96.6%); however, PCV2 was more frequently detected in samples from the offices (37.5%), the farm staff (25%) and the perimeter (21%). These results indicate that PCV2 contamination is frequent in facilities despite the long-term use of vaccination programs. Therefore, PCV2 control programs should include more exhaustive cleaning and disinfection protocols in non-animal facilities, as well as the implementation of specific biosecurity measures in these areas to minimize the risk of PCV2 introduction from external sources.

Zoonotic Enterocytozoon bieneusi genotypes in free-ranging and farmed wild ungulates in Spain.

Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animals.

The fungal-related intracellular parasite Enterocytozoon bieneusi is a worldwide public health and veterinary problem. Here we demonstrated that it was present in wild boar, and wild and farmed red deer in Spain, with genotypes potentially capable of infecting humans, posing a public health risk.

Cardiorespiratory nematodes and co-infections with gastrointestinal parasites in new arrivals at dog and cat shelters in north-western Spain.

Metastrongyloid and trichuroid cardiorespiratory nematodes of dogs and cats are considered emergent in several European countries, and an increase in the number and extent of endemic foci has been described. Since data on their prevalence are limited in this continent, faecal samples from new arrivals (365 dogs and 65 cats) at two animal shelters in North-western Spain were analysed using both floatation and Baermann techniques. In order to confirm the microscopic identification of Metastrongylidae first stage larvae, molecular characterization based on the sequence of the ITS-2 was performed. The possible influence of some variables such as the species, sex and age of the animals and the co-infection with other gastrointestinal parasites on the prevalence of cardiorespiratory nematodes was analysed. The most prevalent metastrongylid was Aelurostrongylus abstrusus (15.4%) followed by Angiostrongylus vasorum (4.1%) and Crenosoma vulpis (1.1%). Regarding trichuroids, Eucoleus aerophilus and/or Eucoleus boehmi eggs were detected in 28 dogs (7.7%) and four cats (6.2%). Almost all animals positive to cardiorespiratory nematodes (86.8%) were co-infected with gastrointestinal parasites. The prevalence of Metastrongylidae and respiratory trichuroids was significantly higher in dogs co-infected with Taenia spp. and Toxocara canis or Giardia duodenalis and Sarcocystis spp., respectively. In cats, a significant higher prevalence of Metastrongylidae nematodes was found in animals co-infected with Toxocara cati. Our results reveal that cardiorespiratory nematodes are common in companion animals from north-western Spain, showing higher prevalences than those previously reported from this country. This investigation represents the first report of C. vulpis, E. aerophilus and E. boehmi in dogs from Spain. The identification of a number of zoonotic parasites is of public health concern. Our results indicate that these nematodes should be included in the differential diagnosis of dogs and cats from north-western Spain showing respiratory or cardiac clinical signs.

A case report of fatal feline babesiosis caused by Babesia canis in north western Spain.

BACKGROUND: In Europe, Babesia infections in cats are sporadic and only partial knowledge is currently available since the number of described cases including both the clinical presentation and the molecular identification of the Babesia species involved is limited. In the present case report, the clinical signs, the epidemiological data and the molecular results suggest that this is the first reported fatal case of feline babesiosis caused by Babesia canis. CASE PRESENTATION: A six month old female European shorthair cat from north-western Spain died after being hospitalized for two days. This animal was pregnant and showed anorexia, lethargy, weakness, jaundice and fever with increased respiratory and heart rates. Haematological analysis revealed haemolytic regenerative anaemia, thrombocytopenia and leukocytosis. The presence of piroplasms was assessed using a PCR targeting the 18S rRNA gene of Babesia spp. and Theileria spp.; the sample resulted positive and B. canis was identified by DNA sequence analysis. The possible existence of co-infections with other vector-borne pathogens such as Anaplasma spp., Bartonella spp., Borrelia burgdorferi s.l., Cytauxzoon spp., Ehrlichia spp., Hepatozoon canis, Mycoplasma spp. or Rickettsia spp. was excluded by qPCR. CONCLUSIONS: Our results together with previous reports on Babesia infections in cats from Europe suggest that feline babesiosis should be included in the differential diagnosis of animals with anaemia, thrombocytopenia, anorexia and lethargy, especially in young or immunocompromised animals from endemic areas for canine babesiosis.

Population genetic structure of the elephant tick Amblyomma tholloni from different elephant populations in Kenya.

Understanding factors that shape tick population genetic structure is important as they may be exploited in crafting strategies for vector control. Amblyomma tholloni, or "elephant tick" is a three-host tick whose adults preferentially parasitize African elephants. The aim of this study was to determine the influence of fragmentation of the host populations on the genetic structure of this tick species from different ecosystems in Kenya, using the second internal transcribed spacer (ITS-2) and mitochondrial cytochrome oxidase 1 (CO1) loci. Population genetic analysis of ticks collected from four elephant populations using ITS-2 and CO1 loci revealed high gene diversity. Gene diversity at the ITS-2 locus was 0.91 and the nucleotide diversity was, 0.01. ITS-2 gene diversity was highest in Laikipia-Samburu ecosystem (0.947) and lowest in Tsavo (0.80). The CO1 locus also had high gene diversity, 0.790, and low nucleotide diversity, 0.006, and like ITS-2, gene diversity was higher in Laikipia-Samburu ecosystem (1.00) and lower in Tsavo (0.286). There was a modest statistically significant genetic differentiation among the four tick populations based on ITS-2 (FST = 0.104, P < 0.001; ΦST = 0.105, P < 0.001), and a 10% of molecular variance attributed to genetic variation between populations. There was also statistically significant differentiation among tick populations using haplotype frequencies for CO1 locus (FST = 0.167, P < 0.001) accounting for 17% of genetic variance among populations, but not modelled genetic distances (ΦST = 0.029, P = 0.095) suggesting very recent genetic differentiation. In addition, populations of A. tholloni in Kenya had a significantly negative Tajima D and Fu & Li's F* and D* at the CO1 locus suggesting recent positive selection. The extensive acaricide use in livestock, which host the larval stage, could be driving purifying selection and genetic hitchhiking of the CO1 locus. However, tests sensitive to demography such as Fu's FS, Ramos-Onsins & Rozas's R2 and raggedness index r were statistically significant at the ITS-2 locus suggesting ancient demographic expansion. Elephant population fragmentation appears to shape the genetic structure of A. tholloni, while agro-ecological factors could influence the genetic diversity of ticks.

Antimicrobial Susceptibility of Enterotoxigenic Escherichia coli from Diarrhoeic Neonatal Calves in Spain.

Enterotoxigenic Escherichia coli (ETEC) is one of the major pathogens involved in neonatal calf diarrhoea (NCD) causing high economic losses in dairy farms. Antibiotic treatment is common in cases of systemic illness caused by NCD, but antimicrobial susceptibility tests (AST) are usually not performed. Thus, the aim of this study was to characterize the antimicrobial susceptibility of ETEC strains obtained from calves with diarrhoea between 2018-2020. Faecal samples (n = 420) were analyzed to detect the typical ETEC virulence factors F5 and STa. Positive samples were cultured to identify and isolate ETEC strains (n = 41) and ASTs were performed. Our results are alarming since ETEC strains resistant to three or more families of antimicrobials were detected in all isolates. Only four antibiotics (ceftiofur, cefoperazone, cefquinome and gentamicin) presented efficacy against more than 90% of the ETEC strains, while the other ten antibiotics were effective against less than 40% of the strains. In addition, a high number of strains were resistant to most first-line antimicrobials used in veterinary practice. For this reason, when ETEC infection is suspected, an AST must always be performed to select the most appropriate antimicrobial in each case and to avoid the emergence of new resistance mechanisms.

Monitoring of porcine circovirus type 2 infection through air and surface samples in vaccinated and unvaccinated fattening farms.

Air and surfaces of swine farms are the two alternative samples to obtain information about the health status of the herd. The aim of this study was to assess air and surface sampling for the detection of porcine circovirus type 2 (PCV2) in vaccinated and unvaccinated fattening farms, studying the relationship between the viral load in these samples with the viremia at herd level. Three swine fattening batches (one unvaccinated; two vaccinated) were monitored at 10, 12, 14, 16 and 18 weeks old; at each stage, blood, air and different surfaces were sampled and analysed by qPCR. In all herds, PCV2 was detected in all types of samples. Whenever viremia was detected, PCV2 was also detected in air and surface samples, even in those cases with a low estimated prevalence (1.6%); moreover, in two out of the three herds, PCV2 was detected in air and surface samples earlier than in the blood of the sampled population. In addition, a good correlation between the viremia of pig population and the PCV2 load in air and surface samples was found in both cases (τ = 0.672 and 0.746, respectively; p <0.05). These results show that air and surface samples could be useful tools to monitor PCV2 infection, being suitable for detecting the virus in cases of low prevalence and even before pigs develop viremia; therefore, these sampling techniques would speed up the implementation of the required measures to prevent productive and economic losses due to PCV2 infection.

Mixed Nasopharyngeal Myiasis by Bots and Blowflies in a Roe Deer (Capreolus capreolus).

A mixed nasopharyngeal infestation by larvae of Cephenemyia stimulator (Diptera: Oestridae) and Lucilia caesar (Diptera: Calliphoridae) was detected in a roe deer (Capreolus capreolus) killed in a car collision in Lugo, Spain, in July 2020. This article stresses the importance of correctly identifying larvae in myiasis cases.

Diversity of Anaplasma species and importance of mixed infections in roe deer from Spain.

Although wildlife can act as reservoirs of some Anaplasma species, studies on the presence and distribution of Anaplasma spp. in wild cervids are mainly limited and focused on zoonotic species. In order to identify the Anaplasma species in roe deer from Spain and to detect co-infections, 224 spleen samples were tested for Anaplasma spp. using a commercial qPCR; positive samples were further characterized using generic 16S rRNA primers and species-specific primers targeting the msp2 and groEL genes. Anaplasma DNA was detected in the 50.9% of samples, and four Anaplasma species were identified. Anaplasma phagocytophilum (43.8%) was predominant, followed by Anaplasma bovis (13.8%), Anaplasma capra (5.8%) and Anaplasma ovis (2.2%). In addition, strains similar to Anaplasma platys were found in nine animals. Most positive roe deer (71.9%) were infected with a single Anaplasma species, whereas co-infections with two (19.3%) or three (8.8%) Anaplasma species were also found. This study confirms the widespread occurrence of Anaplasma spp. in roe deer from Spain, being the first report of A. platys-like strains and A. capra in this cervid; it is also the first report of A. capra in Spain. The detection of Anaplasma species pathogenic for humans and/or domestic animals in roe deer suggests that this cervid may play a role in the sylvatic cycle of these bacteria contributing to the appearance of clinical anaplasmosis cases. In addition, co-infections are common in roe deer revealing that Anaplasma species specific PCR assays are essential for a reliable identification as well as for determining their real prevalence.

Molecular identification of zoonotic Rickettsia species in Ixodidae parasitizing wild lagomorphs from Mediterranean ecosystems.

A survey study was carried out to identify tick species parasitizing wild lagomorphs in Mediterranean ecosystems in southern Spain and to determine the occurrence of Rickettsia species present in these ticks in this region. A total of 1304 European wild rabbits (Oryctolagus cuniculus) and 58 Iberian hares (Lepus granatensis) were individually examined for the presence of ticks. Ticks were found in 42.9% and 50% of the wild rabbits and hares sampled, respectively. A total of 1122 ticks were collected and five species, including Rhipicephalus pusillus, Hyalomma lusitanicum, Haemaphysalis hispanica, Ixodes ventalloi and Rhipicephalus sanguineus sensu lato (s.l.), were microscopically and molecularly identified at the 16S rRNA gene. This is the first study on Ixodidae parasitizing Iberian hares. The presence of Rickettsia DNA was assessed in 254 tick pools (according to hunting states, lagomorph species, tick species and tick development stage) using PCR assays targeting the rOmpA, rOmpB and gltA. Twenty-seven pools (10.6%) were positive to Rickettsia DNA. Five zoonotic Rickettsia species were identified, being Rickettsia massiliae the most frequent (4.7%), followed by Rickettsia sibirica subsp. mongolitimonae (2.8%), Rickettsia slovaca (2.0%), Rickettsia aeschlimannii (0.8%) and Rickettsia africae (0.4%). The results suggest that wild rabbits and Iberian hares are parasitized by a wide range of tick species and that these lagomorphs may play an important role in the sylvatic cycle of some zoonotic Rickettsia species in Mediterranean ecosystems. Our data represent the first report of R. massiliae, R. aeschlimannii, R. slovaca and R africae in ticks collected in wild lagomorphs in Europe, and the first report of not imported R. africae in this continent. Since R. slovaca and R. africae DNA was detected in tick species different to their main vectors, further studies are warranted to unravel the role of wild lagomorphs in the epidemiology of these vector-borne pathogens.

An Update on Cephenemyiosis in the European Roe Deer: Emergent Myiasis in Spain.

Cephenemyia stimulator is a Palearctic species developing in the nasal cavity and pharynx of roe deer (Capreolus capreolus). It is widely spread in the range of distribution of this ungulate in Europe. Since the first report of C. stimulator in Spain in 2001, a rapid geographic expansion has been observed, first in the north of the country, with high prevalence and intensities of infestation that caused some mortal cases, and, lately, also in Extremadura and Andalucía, the southernmost populations of European roe deer. These observations suggest an adaptation of this parasite to different ecosystems of the Iberian Peninsula. Almost simultaneously, C. stimulator is also expanding its range to northern Europe, with the first cases being reported in Sweden. Thus, Cephenemyia stimulator may be an example of a parasite currently displaying distributional changes along its southernmost and northernmost range margins. Thus, it is of the utmost importance to unravel all the epidemiological and clinical aspects of this myiasis, as well as implementing surveillance measures including reliable and non-invasive diagnostic techniques to monitor its expansion and adaptation to different ecosystems and/or hosts and to reduce the negative impact on roe deer populations.

Spatiotemporal monitoring of Cysticercus pisiformis in European wild rabbit (Oryctolagus cuniculus) in Mediterranean ecosystems in southern Spain.

Cysticercosis in wild lagomorphs is caused by Cysticercus pisiformis, the larval stage of Taenia pisiformis. Although previous studies have reported the presence of T. pisiformis in different wild carnivore species, information about the prevalence of C. pisiformis in their intermediate hosts is still very scarce. An epidemiological surveillance program was carried out to determine the prevalence and spatiotemporal patterns of cysticercosis in wild rabbits (Oryctolagus cuniculus) from Spanish Mediterranean ecosystems. A total of 2,923 animals were sampled in 164 hunting estates from Andalusia (southern Spain) during four study periods: 2009-2012 (P1), 2012-2015 (P2), 2015-2018 (P3) and 2018-2020 (P4). The presence of cysticerci was assessed by macroscopical examination and a subset of the collected parasites were molecularly identified by conventional PCR targeting the ITS-1 and 12S rRNA partial genes of Taenia spp. Risk factors associated with cysticercus infection were assessed by generalized estimating equation (GEE) analysis. A spatial statistical analysis was carried out using a Bernoulli model to identify statistically significant spatial clusters. Cysticercus infection was confirmed in 81 (2.8 %; 95 % CI: 2.2-3.4) rabbits. Cysticerci from 18 infected animals were molecularly identified as T. pisiformis. The GEE model showed the study period as the only risk factor associated with C. pisiformis infection in wild rabbits. Significantly higher prevalence was found in P2 (6.1 %; 95 % CI: 4.4-8.4) compared to the rest of the periods. At least one cysticerci-positive animal was detected in 41 (25.0 %; 95 % CI: 18.4-31.6) out of the 164 hunting estates. No statistically significant spatial clusters of high cysticercus prevalence were identified. Our results indicate an endemic circulation of C. pisiformis in wild rabbits in southern Spain. The spatial results highlight a widespread distribution of this parasite in their populations. Further studies should focus in determining which sympatric species may act as definitive hosts for T. pisiformis and the relevance of other potential intermediate host species (e.g. hares and rodents), as the relevance of wild rabbits in the sylvatic cycle of this cestode in Mediterranean ecosystems seems to be low.

The Goat as a Risk Factor for Parasitic Infections in Ovine Flocks.

Small ruminants in northwestern Spain are frequently managed in mixed flocks. Sheep-goat joint management has not been considered a risk factor for parasite infection, so the main objective of this study was to establish if mixed management with goats supposes a risk factor for parasitic infections in ovine flocks. Two thousand and ninety-three sheep were sampled from 74 commercial meat ovine flocks for diagnostic of the main parasites. Goat contact was a risk factor for sheep to be infected by protostrongylids, Dictyocaulus filaria, gastrointestinal nematodes and Eimeria spp. In contrast, Moniezia, Fasciola hepatica, Dicrocoelium dendriticum and Paramphistomidae prevalences were not influenced. Sheep-goat mixed management can be considered as a risk factor, since goats would act as a source of pasture contamination for interspecific parasites (protostrongylids, Dictyocaulus filaria and gastrointestinal nematodes). In relation to host-specific parasites, such as Eimeria spp., goats cannot be considered as a source for sheep, but competition for food and spaces between both small ungulates can suppose a reduced grazing area to sheep, provoking high environmental contamination and stress that facilitate their infection. Future epidemiological studies for parasitic infections in small ruminants should consider sheep-goat mixed management as a possible risk factor to be included in multivariate analyses.

Efficacy of Two Commercial Ready-To-Use PCV2 and Mycoplasma hyopneumoniae Vaccines under Field Conditions.

Porcine Circovirus Type 2 (PCV2) and Mycoplasma hyopneumoniae are economically important pathogens in swine farms. Vaccination is the main preventive measure for both infections. In order to test two ready-to-use bivalent vaccines, 646 piglets from a herd actively infected with both pathogens were stratified according to the sow parity number and randomly assigned to three groups: A and B were vaccinated with two different vaccines, respectively, while C remained as the unvaccinated control. Vaccine efficacy was assessed based on the weight, average daily weight gain (ADWG), degree of lung lesions, presence of PCV2 viremia by qPCR and presence of PCV2 and M. hyopneumoniae antibody levels by ELISA. Our data revealed that the sow parity did not influence the vaccine outcomes. Good results for most of the analyzed parameters were observed in both vaccinated groups. ADGW and final weight were higher and lung lesions were less evident in both vaccinated groups than in the control one, but only Group A showed a significant improvement. PCV2 viremia was not detected in Group A, but it did appear in Group B coinciding with its peak in Group C. Finally, both the PCV2 and M. hyopneumoniae serological patterns differed depending on the employed vaccine.

Ectoparasites of wild rabbit (Oryctolagus cuniculus) in Southern Italy.

The European wild rabbit Oryctolagus cuniculus is a widespread mammal that may act as host for ectoparasites and reservoir for some vector-borne pathogens. Study aims were to investigate the arthropod fauna that may infect the European wild rabbit, to assess the ecological indexes and to investigate tick preferred attachment sites. The survey was conducted on 105 wild rabbits hunted by poachers in a Mediterranean woodland area in the province of Ragusa. Animals were individually examined for ectoparasite presence and the tick load assessed on eleven body anatomical sites. Ticks and fleas were found on 79 (75.2%) and 3 (2.9%) out of the 105 examined rabbits, respectively. Three tick species were identified; Rhipicephalus pusillus was the most abundant followed by Rhipicephalus turanicus and Ixodes gibbosus. Although ticks were found on all anatomical sites, ears, forelimbs, and abdomen were those more frequently infected. All the collected fleas were identified as Spilopsyllus cuniculi. Some of the ectoparasite species found on examined wild rabbits are recognized as potential vectors of pathogens of animal and human concern, such as, Rickettsia sibirica mongolitimonae and Rickettsia massiliae. The most abundant tick species found in this study is the main vector of some zoonotic pathogens; therefore, the possible role of O. cuniculus in their maintenance should be further investigated.