Characterization of biosecurity practices among cattle transport drivers in Spain.

Transmission of pathogens between farms via animal transport vehicles is a potential concern; however, the available information on driver routines and biosecurity measures implemented during transport is limited. Given the above, the aim of this study was to describe and characterize the prevailing practices and biosecurity measures adopted by cattle transport drivers in Spain. Eighty-two drivers were surveyed via face-to-face or remotely. The survey included questions on general characteristics of the drivers (type of journeys and vehicles) together with biosecurity practices implemented during cattle transport and vehicle hygiene practices. Results showed that several risky practices are performed quite frequently such as visiting different premises with different levels of risk (e.g., breeder and fattening farms); entering the farm premises to load/unload animals, passing by several farms to load and unload animals, or not always cleaning and disinfecting the vehicle between travels, among others. To explore similarities among the drivers and identify groups sharing specific practices, hierarchical clustering on principal components (HCPC) was computed on the results of multiple correspondence analysis (MCA). The first three MCA dimensions (out of 13) were retained in the agglomerative clustering and four different clusters were identified. Clusters 1 and Cluster 4 accounted for 39.5% and 29.6% of respondents, respectively. The clusters were mainly differentiated by practices in the loading/unloading of cattle, such as the frequency of contact with animals remaining on the farm, and the frequency of the vehicle's disinfection between farms. Cluster 2 and Cluster 3 were of similar size, about 15% of respondents each. Cluster 2 consisted of drivers who mainly made journeys to slaughterhouse, while drivers in Cluster 3 were characterised by the use of working clothes and boots. Based on these findings, it is advisable to increase awareness on the role that animal transport can have in the spread of pathogens between cattle farms and the importance of biosecurity in preventing such transmission. There is also a need to support animal transport professionals in such task, not only through the development of initiatives to increase awareness, but also through the investment in improving cleaning and disinfection facilities and to consider the economic cost associated with some practices to not compromise the economic viability of the sector.

Intestinal Parasites and Fecal Cortisol Metabolites in Multi-Unowned-Cat Environments: The Impact of Housing Conditions.

Housing conditions were assessed in different unowned multi-cat management models in order to evaluate their impact on the occurrence of intestinal parasites and fecal cortisol metabolite (FCM) levels. Fresh stool fecal samples were collected from rescue shelters, catteries and feline colonies for coprological analyses in order to detect intestinal parasite patency and fecal cortisol metabolites. A questionnaire provided information about the facilities, management and housing conditions of cats, including information about dog exposure, enclosure size, environment enrichment and changes to group composition. Overall, intestinal parasite infection was detected in 58.2% of fecal samples collected. The occurrence of intestinal parasites detected in free-roaming cats was 82.2%, mainly due to helminth infection. The parasite infection rate was 57.3% in rescue shelters and 34.6% in catteries. In confined cats, protozoa infection was more likely detected in rescue shelters than in catteries (RR = 2.02 (1.30-3.14), p = 0.0012). Although the FCM values were very variable between cats, the enclosure size and parasite infection were correlated with the average FCM. A small enclosure size was correlated with high fecal cortisol metabolites (p = 0.016). Protozoa-positive samples showed higher FCM levels than negative samples (p = 0.0150). High dog exposure was statistically associated with protozoa infection (p = 0.0006). The results indicated that improving housing, especially in terms of floor space and avoiding dog exposure, reduces stress and can thus be applied to make control strategies in multi-unowned-cat environments more efficient, especially when cats are confined.

Detection of Rickettsia massiliae/Bar29 and Rickettsia conorii in red foxes (Vulpes vulpes) and their Rhipicephalus sanguineus complex ticks.

To determine the prevalence of exposure to Rickettsia massiliae/Bar29 and Rickettsia conorii in wild red foxes, we collected blood samples and ticks from 135 foxes shot in different game reserve areas in Catalonia. To detect SFG rickettsia in Rhipicephalus sanguineus complex ticks collected from the foxes, we used real-time polymerase chain reaction (PCR) to screen for ompA gene and a tick-borne bacteria flow chip technique based on multiplex PCR. Serum samples were positive for antibodies against spotted fever group (SFG) rickettsiae in 68 (50.3%). Molecular techniques identified R. massiliae in 107 ticks, R. aeschlimannii in 3 ticks, and R. slovaca in one tick; no R. conorii was identified in any of the ticks analyzed. We conclude that red foxes can carry ticks with SFG rickettsia.

On the possible role of ticks in the eco-epidemiology of Coxiella burnetii in a Mediterranean ecosystem.

Ruminant livestock is the main reservoir of Coxiella burnetii (Cb), but little is known about the role of wildlife and ticks in its epidemiology. The Iberian ibex (Capra pyrenaica, Schinz 1838) population of "Ports de Tortosa i Beseit" (NE Spain) suffers intense tick infestations and low reproduction rates. This study aims to (1) assess the relationship between infection in ibexes (detection of serum antibodies and/or of Cb DNA in tissues) and Cb DNA presence in ticks hosted by the same ibexes; and (2) identify Cb associated risk factors. Between 2011 and 2015, serum (n = 130), spleen (n = 72), lymph node (n = 89) and tick (n = 669) samples from 134 hunter-harvested ibexes were collected. Antibody detection was performed by ELISA and Cb DNA presence was assessed by PCR. Potential risk factors were assessed with regression tree models. Although 30% of the ibexes (39/130; 95%CI, [10%-29.8%]) had antibodies, Cb DNA was detected in only 9.8% of the ibexes (11/112; 95%CI [7.6%-27.25%]). The prevalence of Cb-carrier ticks averaged 10% and exceeded 20% for the genus Haemaphysalis. However, lacking correlation between infection in ibexes and their ticks does not support tick-to-ibex transmission or vice versa. Tree modelling points to host, population and environmental factors as drivers of Cb infection in ticks and suggests connections with the domestic cycle. The percentage of Cb-carrier ticks detected is noteworthy. Along with heavy tick infestations, it suggests vector potential for these tick species, especially for the genera Rhipicephalus and Haemaphysalis. Since vector competence has not been assessed in these tick species, a classic vector role cannot be proposed nor discarded, but promoter factors of vector capacity occur. In addition, the risk of tick-borne infection through tick excreta should not be neglected. While the airborne route is the preeminent route for Cb infection, ticks' contribution to Cb epidemiology deserves further attention.

Cardiopulmonary helminths in foxes from the Pyrenees.

The present survey was carried out to investigate the prevalence of cardiopulmonary helminths in red foxes in Pyrenees area and to evaluate the role of foxes in the eco-epidemiology of these nematodes. Hearts and entire respiratory tracts were obtained from 87 foxes from Vall d'Aran region, Pyrenees, Catalonia, north-eastern Spain. The cardiopulmonary tracts were dissected, flushed and examined for nematodes using sedimented flushing water. Of the 87 examined foxes, 53 (61%) were positive for cardiopulmonary helminths. The identified nematodes were Crenosoma vulpis (44.8%), Eucoleus aerophilus (29.9%) and Angiostrongylus vasorum (3.4%). Statistical differences were observed only on comparing age and C.vulpis prevalence, with young foxes being more infected than adults. The high prevalence of cardiopulmonary nematodes suggested that red foxes may play an important role in their transmission and maintenance in the studied area.

Reverse line blot hybridization used to identify hemoprotozoa in Minorcan cattle.

Piroplasmosis, a tick-borne protozoal disease, is an important disease affecting domestic and wild animals. We performed PCR-based reverse line blot hybridization (RLB) assays on blood samples obtained from 133 cattle exposed to ticks in field conditions in Minorca (Balearic Islands, Spain) in three different seasons. The oligonucleotides used were those for Theileria annulata, T. buffeli, T. taurotragi, T. velifera, Babesia bigemina, B. bovis, B. divergens, and B. major. The RLB technique allowed the simultaneous identification of T. annulata, T. buffeli, B. bigemina, and B. bovis as the piroplasms present in cattle in Minorca. Of the 133 animals, only 4 were not infected by any of the studied parasites. The results indicated endemic piroplasm infection in cattle in Minorca; especially important was the presence of T. annulata. The RLB was highly sensitive and allowed the simultaneous detection and identification of the Theileria and Babesia species in carrier cattle, which cannot be achieved by classical identification methods.

Pan-Mediterranean comparison for the molecular detection of Theileria annulata.

Seven laboratories decided to compare their molecular diagnostic techniques to identify Mediterranean theileriosis caused by Theileria annulata. Each laboratory used either PCR or PCR and reverse line blot hybridization (RLB) to identify T. annulata. Five laboratories sent their own samples to laboratory 4 to be recoded and passed on to at least two other laboratories. A total of 120 blood samples were analyzed during this study, generating 540 results. Laboratory 1 sent only T. annulata-infected samples (positive control batch), and all the laboratories testing this batch found 100% infection. Laboratory 2 sent only negative samples from a Mediterranean area where T. annulata was unknown, and two laboratories out of three found a few positive samples in these negative samples. For the remaining samples, detection performance was variable. Agreement between laboratories ranged from 21.4 to 91.3%. The overall mismatch between laboratories was around 30% by whatever technique used. This paper describes the methodological parameters that could explain the variation of results.

Seroprevalence of antibodies to Neospora caninum in dogs from Spain.

The prevalence of Neospora caninum antibodies was determined in sera of 139 dogs from Catalonia (northeastern Spain) using the indirect immunofluorescence antibody test (IFAT). Antibodies in the IFAT were found in 17 of 139 dogs (12.2%) with titers ranging from 1:50 to 1: 1,600. Seroprevalence was higher in dogs over 1 yr old compared with dogs younger than 1 yr (P < 0.05). No statistical difference was observed when sex, breed, purpose, or modus vivendi was compared with seropositivity. Most dogs had low antibody titers, which indicated subclinical infection in the area studied. No neosporosis-related disease was reported from any dog, although a German shepherd with an antibody titer of 1:800 showed pododermatitis. All sera were also screened using a commercial direct agglutination test (DAT). The DAT showed a similar specificity but a lower sensitivity when compared with IFAT as a reference technique.

Parasitic helminths in the digestive tract of six species of owls (Strigiformes) in Spain.

A survey was carried out in Catalonia, Spain to determine the prevalence of parasitic helminths infesting the digestive tract of nocturnal raptors (Strigiformes). One hundred birds belonging to six owl species were examined. The number of birds examined ranged between three and 30. The overall level of infestation was 65% and the most frequent helminths present were nematodes, followed by trematodes, cestodes and acanthocephalans. Strigiformes showing the highest levels of infestation were little owls (Athene noctua), 86.7% of which were infested. Helminths with the highest prevalence were Synhimantus spp., intestinal capillarids, Brachylaima spp., cestodes and Centrorhynchus aluconis.

Prevalence of intestinal parasites in shelter and hunting dogs in Catalonia, Northeastern Spain.

To compare the prevalence of intestinal parasites in shelter and hunting dogs in Catalonia, Northeastern Spain, fresh faecal samples from 81 shelter dogs and 88 hunting dogs were collected and analysed by faecal flotation. The overall prevalence of intestinal parasites was 71.6% in each population. In the shelter dog group, 67.9% of dogs were positive for intestinal protozoa and 9.8% were positive for helminths. In the hunting dog group, 20.4% of dogs were positive for intestinal protozoa and 63.6% were positive for helminths. A subset of Giardia-positive samples was evaluated by PCR; Giardia assemblages C or D were detected. These results suggest that comprehensive parasite control measures should be implemented in both shelter and hunting dogs in Catalonia.

Molecular detection of Rickettsia typhi in cats and fleas.

BACKGROUND: Rickettsiatyphi is the etiological agent of murine typhus (MT), a disease transmitted by two cycles: rat-flea-rat, and peridomestic cycle. Murine typhus is often misdiagnosed and underreported. A correct diagnosis is important because MT can cause severe illness and death. Our previous seroprevalence results pointed to presence of human R. typhi infection in our region; however, no clinical case has been reported. Although cats have been related to MT, no naturally infected cat has been described. The aim of the study is to confirm the existence of R. typhi in our location analyzing its presence in cats and fleas. METHODOLOGY/PRINCIPAL FINDINGS: 221 cats and 80 fleas were collected from Veterinary clinics, shelters, and the street (2001-2009). Variables surveyed were: date of collection, age, sex, municipality, living place, outdoor activities, demographic area, healthy status, contact with animals, and ectoparasite infestation. IgG against R. typhi were evaluated by indirect immunofluorescence assay. Molecular detection in cats and fleas was performed by real-time PCR. Cultures were performed in those cats with positive molecular detection. Statistical analysis was carried out using SPSS. A p < 0.05 was considered significant. Thirty-five (15.8%) cats were seropositive. There were no significant associations among seropositivity and any variables. R. typhi was detected in 5 blood and 2 cultures. High titres and molecular detection were observed in stray cats and pets, as well as in spring and winter. All fleas were Ctenocephalides felis. R. typhi was detected in 44 fleas (55%), from shelters and pets. Co-infection with R. felis was observed. CONCLUSIONS: Although no clinical case has been described in this area, the presence of R. typhi in cats and fleas is demonstrated. Moreover, a considerable percentage of those animals lived in households. To our knowledge, this is the first time R. typhi is detected in naturally infected cats.

Evaluation of the presence of Rickettsia slovaca infection in domestic ruminants in Catalonia, Northeastern Spain.

Rickettsia slovaca is the etiological agent of the human disease tick-borne lymphadenopathy (TIBOLA) transmitted by Dermacentor spp. ticks. In our area, Dermacentor marginatus is the most important tick vector; adult ticks feed on mammals, especially ungulates such as wild boars and domestic ruminants. The epidemiology of tick-transmitted diseases describes a wild cycle and a domestic cycle and both are connected by ticks. To identify the role of domestic ruminants in the transmission and maintenance of R. slovaca infection, blood samples from sheep (n=95), goats (n=91), and bullfighting cattle (n=100) were collected during a herd health program, and livestock grazing was selected to ensure tick contact. Samples were analyzed by serology using an indirect immunofluorescent assay (IFA) and molecular techniques (real-time PCR). Seroprevalence was 15.7% in sheep, 20.8% in goats, and 65.0% in bullfighting cattle. On the basis of molecular methods, R. slovaca infection was demonstrated in a goat blood sample with an antibody titer of 1:160. This is the first time that R. slovaca has been identified in a goat blood sample. These results suggest that domestic ruminants are exposed to R. slovaca infection and, because the domestic cycle is close to the human environment, this could increase the risk of transmitting the pathogen to human beings.

A carboxypeptidase inhibitor from the tick Rhipicephalus bursa: isolation, cDNA cloning, recombinant expression, and characterization.

A novel proteinaceous metallo-carboxypeptidase inhibitor, named tick carboxypeptidase inhibitor (TCI), was isolated from the ixodid tick Rhipicephalus bursa and N-terminally sequenced. The complete cDNA encoding this protein was cloned from tick mRNA by reverse transcription-PCR and rapid amplification of cDNA ends techniques. The full-length TCI cDNA contains an open reading frame coding for a precursor protein of 97 amino acid residues that consists of a predicted signal peptide of 22 residues and of mature TCI, a 75-residue cysteine-rich protein (12 Cys). The deduced amino acid sequence shows no homology to other known proteins; the C terminus, however, resembles those of other protein metallo-carboxypeptidase inhibitors, suggesting a common mechanism of inhibition. Recombinant TCI expressed in Escherichia coli is fully functional and inhibits carboxypeptidases of the A/B subfamily with equilibrium dissociation constants in the nanomolar range. Structural analyses by circular dichroism and nuclear magnetic resonance indicate that TCI is a protein strongly constrained by disulfide bonds, unusually stable over a wide pH range and highly resistant to denaturing conditions. As a tight binding inhibitor of plasma carboxypeptidase B, also known as thrombin-activatable fibrinolysis inhibitor, recombinant TCI stimulates fibrinolysis in vitro and thus may have potential for applications to prevent or treat thrombotic disorders.