Anna Ortuño

Bovine piroplasms in Minorca (Balearic Islands, Spain): a comparison of PCR-based and light microscopy detection.

The present study provides the first epidemiological data regarding infection by Theileria and Babesia piroplasms in cattle in Minorca. More than 94% of the studied animals were positive for the presence of Theileria sp., and of those, 41.3% were positive for the presence of Theileria annulata. These results indicate that the prevalence of Mediterranean theileriosis caused by T. annulata is very high in Minorcan dairy farms and that other Theileria sp. are also present in the area. The prevalence of infection was similar throughout the study indicating an endemic situation in this island. The use of PCR resulted in significantly higher efficacy of detection of Theileria sp. compared to microscopical observation (MO) of blood smears and allowed the specific discrimination between pathogenic and non-pathogenic theilerias which cannot be accomplished by traditional diagnosis by MO. Babesia infection in the area was mainly due to Babesia bigemina (6.0% of the studied animals were infected), while one animal (0.75%) was found to be infected by Babesia bovis. It was observed that 31% of animals infected with B. bigemina had a concurrent infection of T. annulata. PCR also resulted in a significantly higher efficacy of detection of Babesia sp. compared to MO when infection levels were higher, towards the end of the study period. The results clearly demonstrate that parasitic infection by piroplasms, especially Theileria sp. is common and endemic in the island of Minorca and that PCR is the optimal approach for the detection and discrimination of these important parasites.

Anti-HEV antibodies in domestic animal species and rodents from Spain using a genotype 3-based ELISA.

A truncated ORF2 capsid HEV antigen derived from a genotype 3 strain was developed in insect cells and insect larvae, and compared with the Sar55 antigen and a commercial ELISA. The antigen expressed in insect cells showed a better correlation with Sar55 (kappa value (k)=0.84) than the insect larvae antigen (k=0.69), and a better reproducibility as indicated by the intra and interplate variation coefficients. Commercial ELISA designed for human diagnosis but adapted to animal use using specific secondary antibodies demonstrated to have a very low sensitivity. The insect cell expressed antigen was used to develop an ELISA to detect anti-HEV-IgG in serum samples of different domestic animal and rodents. Seropositivity in the studied animal populations was 71.4% for pigs, 0.60% for goats, 1.92% for sheep, and 11.11% for cats. None of the 1170 cattle samples or 166 rodent samples analyzed was positive.

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.

A Survey of Ticks (Acari: Ixodidae) on Dairy Cattle on the Island of Menorca in Spain

A survey of ticks affecting dairy cattle under extensive management on Menorca island (off the Spanish Mediterranean coast) was performed through 1999–2000 on seven farms. The species collected were Rhipicephalus bursa, R. turanicus, Hyalomma m. marginatum, H. lusitanicum and Boophilus annulatus. The most abundant species were R. bursa and H. m. marginatum, with peaks of 10.7 (in summer) and 7.5 (in spring) ticks/cow, respectively. R. turanicus was scarcely encountered, while B. annulatus and H. lusitanicum were abundant only in a few localities. The mild climate and adequate vegetation on the island provide satisfactory habitat for extended periods of tick activity, as compared with other regions where these ticks have been collected. The competence of the ticks as vectors for pathogens and the difficulty of using acaricide treatments in the region are discussed.

The role of cats in the eco-epidemiology of spotted fever group diseases

BackgroundMediterranean Spotted Fever (MSF), whose etiological agent is R. conorii, is one of the oldest described vector-borne infectious diseases. Although it is endemic in the Mediterranean area, clinical cases have also been reported in other regions. R. massiliae- Bar29 is related to MSF cases. This strain is distributed worldwide. R. conorii and R. massiliae- Bar29 are transmitted by ticks. Dogs are considered the sentinel of R. conorii infection. Cats could also be involved in their transmission. Rickettsia felis, etiological agent of Flea-borne spotted fever, is mainly transmitted by the cat flea, Ctenocephalides felis. Up to now, the role of cats in its transmission is not entirely elucidated. The aim of the study is to analyze the infection in cats by these microorganisms.MethodsThe study was undertaken in Northeastern Spain. Twenty municipalities of seven regions participated in the study. 212 cats (pets and stray cats) were analyzed. Variables surveyed were: date of collection, age, sex, municipality, source, living place, outdoor activities, health status, type of disease, contact with other animals, and ectoparasite infestation. Sera were evaluated by indirect immunofluorescence antibody assay (IFA). Molecular detection (real-time PCR and sequencing) and cultures were performed on blood samples.ResultsThere were 59 (27.8%) cats seroreactive to one or more microorganisms. Considering cross-reactions, the seroprevalences were 15.6%-19.5% (R. massiliae-Bar29), 1.9%-6.2% (R. conorii), and 5.2%-7.5% (R. felis). A weak association was observed between SFG seropositivity and tick infestation. Ticks found on seropositive cats were Rhipicephalus pusillus, R. sanguineus and R. turanicus. DNA of Rickettsia was detected in 23 cats. 21 of them could be sequenced. Sequences obtained were identical to those sequences of SFG rickettsiae similar to R. conorii and R. massiliae. No amplification of R. felis was obtained.ConclusionsCats can be infected by SFG rickettsiae and produce antibodies against them. Cats may play a role in the transmission cycle of R. conorii and R. massiliae-Bar29, although the role in the R. felis cycle needs further analysis.

Seroprevalence of Rickettsia typhi and Rickettsia felis in dogs from north-eastern Spain.

Rickettsia typhi is the aetiological agent of murine typhus (MT). This disease is often acute and mild, although some fatal cases have been observed [1]. In Spain, clinical cases and serological evidence of exposure to R. typhi in humans have been described [2,3]. In the past, MT has been associated with rodents and rat fleas. Later, a peridomestic animal cycle involving cats, dogs, opossums and their fleas [1] was described. In Spain, R. typhi seropositivity has been shown in dogs [4] from the central region. Rickettsia felis, transmitted by fleas, produces flea-borne spotted fever, a clinical syndrome indistinguishable from MT. Serological evidence of R. felis infection in humans has been observed in Spain [2,3]. R. felis has been detected in fleas commonly found on domestic cats and dogs in Spain [5]. Prevention of these infections depends on recognition of routes of transmission, risk factors for infection, and reservoirs. The aim of this study was to determine the prevalence of both rickettsiae in dog populations from the north-east of Spain.

The dog as an epidemiological marker of Rickettsia conorii infection.

Department of Medicine, Universitat Auto`noma de Barcelona(UAB), Barcelona, SpainINTRODUCTIONDogs are considered to be the sentinels of Rick-ettsia conorii infection, which is the causative agentof Mediterranean spotted fever, one of the mostprevalent tick-transmitted disease in our area. It istransmitted by the dog tick, Rhipicephalus sanguin-eus. This tick is relatively host-specific, and rarelyfeeds on people unless its preferred host—thedog—is not available [1]. This tick is active fromspring to autumn, but climatic changes influenceits activity and, consequently, Mediterraneanspotted fever epidemiology [2].As canine serology constitutes a useful epide-miological marker [3], the objective of this studywas to evaluate the presence of R. conorii in dogpopulations subjected to different exposure toR. sanguineus. For this purpose, three seroepide-miological studies were designed: serosurvey indogs with close contact with ticks, seasonaldifferences in antibodies to R. conorii in dogssubjected to natural tick infestation, and a com-parison of canine seroprevalence rates obtained in1997 and in 2007 in dogs regularly treated forectoparasites.MATERIALS AND METHODS

The role of dogs in the eco-epidemiology of Rickettsia typhi, etiological agent of Murine typhus

Rickettsia typhi, etiological agent of Murine typhus (MT), is transmitted to humans from an animal reservoir through two cycles: a classic rat-flea-rat cycle, and a peridomestic animal cycle. There are not many studies concerning which animals are involved in the peridomestic cycle, and most of them are focused on cats. The aim of this study was to determine the presence of R. typhi in dogs, not only by serological methods but also by direct methods such as culture and molecular detection. Two hundred and one dog blood samples were collected from Veterinary clinics, kennels, and shelters in Northeastern Spain (2006-2008). Age, sex, municipality, living place, healthy status, contact with animals, and ectoparasite infestations were surveyed. IgG was measured by IFA. Titers ≥ 1/64 were considered positive. Cultures were carried out using samples of dogs with titers ≥ 1/128. The molecular detection was performed by real-time PCR. Nine dogs (4.5%) were positive according to IFA (5: 1/64; 3: 1/128; 1: 1/512). There were no significant differences in the rates of antibodies related to any of the variables. Rickettsial DNA was detected in two cultures. Sequences obtained were identical to those of R. typhi. The results show direct and indirect evidences of the presence of R. typhi infection in dogs.

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.

Prevalence of Rickettsia slovaca in Dermacentor marginatus Ticks Removed from Wild Boar (Sus scrofa) in Northeastern Spain

Abstract:  Rickettsia slovaca, the causative agent of TIBOLA, is transmitted by Dermacentor ticks. Dermacentor marginatus is the most widely species distributed in northeasler Spain, and the wild boar constitutes the main host. D. marginatus ticks were collected from hunterkilled wild boar and were tested by PCR/RFLP. Rickettsial DNA–positive ticks were sequenced using the ompA PCR primers. The prevalence of R. slovaca in D. marginatus ticks was 17.7%. Other spotted fever group rickettsiae were detected in ticks, but these were not definitely identified.