Joaquim Castellà

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.

Red foxes (Vulpes vulpes) are a natural intermediate host of Neospora caninum

The present study was undertaken to determine if red foxes are natural intermediate and/or definitive host for Neospora caninum and to study the importance of infection of N. caninum in this species in North-eastern Spain. Faecal samples and brain tissues were obtained from 122 foxes from 21 rural areas of Catalonia. Faeces collected were examined for parasite eggs and coccidian oocysts using sucrose flotation. For PCR-based diagnosis of N. caninum in brain tissues, the specific genomic Nc5 region was selected as the target sequence for DNA amplification. To control for PCR failure and facilitate identification of truly negative samples, the competitor pNc5C molecule was added to all negative samples in a second round of PCR reactions. Of the 122 foxes analysed, 13 (10.7%) were positive by PCR for N. caninum. Signal intensities of all positive samples were relatively weak with the exception of one sample from a 3-month male animal, that also showed the highest repeatability. No differences were observed by sex, age or area of sampling analysis. Detection of stages of N. caninum in brain from naturally infected red foxes demonstrated that red foxes are a natural intermediate host for N. caninum. Faecal samples were analysed for the presence of N. caninum oocysts, however, no oocysts compatible with N. caninum were found. A widespread latent infection of red foxes in North-eastern Spain found in the present study indicates that red foxes could have a very important role in the epidemiology of neosporosis in our area.

Borrelia burgdorferi sensu lato in ticks (Acari: Ixodidae) from two different foci in Spain

The prevalence of Borrelia burgdorferi sensu lato in several tick species was studied over a 2 year period in two ecologically different areas in Spain. One area was an endemic area for Lyme disease, with a number of autochthonous human cases and supported large populations of Ixodes ricinus on cattle and birds; the second area was characterized by the absence of I. ricinus together with the presence of foxes and their associated tick species. While I. ricinus was the main vector of B. burgdoreri in the endemic area (with a mean prevalence of 14% in adults and 51% in nymphs), adults of both Ixodes canisuga and Ixodes hexagonus had high rates of B. burgdorferi prevalence (30 and 28%, respectively) in the zone where I. ricinus was absent. Immatures of Ixodes frontalis were found to be carriers of the spirochete only in those zones where I. ricinus is present, suggesting evidence for reservoir competence in a tick-bird cycle.

Detection and monitoring of mosquito flaviviruses in Spain between 2001 and 2005.

Between the years 2001 and 2005, a total of 72,895 female mosquitoes were trapped during their season of abundance, and analyzed. They were sorted into 4,723 pools belonging to 20 Culicidae species from the Anopheles, Aedes, Ochlerotatus, Culex, Culiseta, Coquillettidia, and Uranotaenia genera. The aim was to detect arboviral RNA directly from mosquito homogenates for the genera Alphavirus, Flavivirus, and Phlebovirus. The study formed part of general arbovirus transmission research in four of the most important wetlands in Spain; in the provinces of Girona, Barcelona, Tarragona, and Huelva. The mosquitoes were collected using human bait, CO(2) traps, or light traps, and they were pooled according to date of collection, location, and species. No arboviral RNA from known pathogenic arboviruses was found. However, 111 pools tested positive for unknown mosquito Flavivirus, the only genus detected. The Flavivirus sequences identified were different from all known Flavivirus mosquito viruses, but very close to Kamiti River virus or cell fusing agent virus. The maximum likelihood estimation infection rate (MLE) was calculated for all regions and species. Aedes albopictus had the highest MLE at 47.14, followed by Ae. vexans with 43.67 (over the entire area). These species were followed by Culiseta annulata, with 36.00. The most common species, Ochlerotatus caspius and Culex pipiens, had low MLE values-0.94 and 0.38, respectively-over the area as a whole.

Canine filariasis. Importance and transmission in the Baix Llobregat area, Barcelona (Spain).

Field and laboratory studies were performed in order to assess the degree of canine dirofilariasis caused by Dirofilaria immitis (Leidy) in the Baix Llobregat region, a fluvial area near Barcelona, Spain. A total of 188 dogs were sampled between May and August of 1994. Three main areas were chosen: the Western Delta, the Eastern Delta and the Northern zone. Simultaneously, a mosquito sampling programme was carried out with CO2 light traps, to search for infective larvae (L3) of D. immitis. Of the 188 dogs sampled, 38 were positive for at least one of the three filaria found: D. immitis 12.8%, Dipetalonema reconditum (Grassi) 3.7% and Dipetalonema dracunculoides (Cobbold) 2.7%. Only 1.1% showed a mixed infection of both D. immitis and D. dracunculoides. Although Dirofilaria repens Raillet et Henry has been found in Spain, it was not found in this study. Comparing the three zones of the Baix Llobregat, the Eastern Delta showed the highest level of D. immitis (35.3%), probably due to the presence of Aedes caspius (Pallas). Despite the effort in sampling the mosquito population, D. immitis was not found in any of the 2001 females dissected, belonging to 5 species.

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.

In vivo and in vitro sensitivity of Trichomonas gallinae to some nitroimidazole drugs

A study was carried out in order to establish the in vivo efficacy of two nitroimidazole drugs against the flagellate Trichomonas gallinae. The results obtained showed a high proportion of therapeutic failures. Thus, carnidazole and dimetridazole failed to eliminate infection in 13 of 17 and 20 of 21 racing pigeons, respectively. The in vitro susceptibility of four T. gallinae isolates to five nitroimidazolic drugs, that is, carnidazole, dimetridazole, metronidazole, ornidazole and ronidazole, was also determined. Minimal lethal concentrations (MLCs) of these drugs were obtained. One of the isolates tested had already proved to be resistant in the first in vivo tests, while the other three had been obtained from wild birds. We were able to confirm in vitro the resistance of the racing pigeon isolate to all the nitroimidazole drugs tested. For carnidazole, dimetridazole, metronidazole and ornidazole the MLC ranged between 93.75-500 microg ml. Although ronidazole showed a greater potency than the other nitroimidazole derivatives against this isolate (MLC: 15.62-31.25 microg ml), 8-22 times more drug was necessary for efficacy against this isolate compared to the others. These other three parasite isolates proved to be sensitive to the five drugs tested (MLC: 0.97-7.81 microg ml).

Isolation of the trichomonad Tetratrichomonas buttreyi (Hibler et al., 1960) Honigberg, 1963 in bovine diarrhoeic faeces

A trichomonad was found in the faeces of a heifer with watery diarrhoea. It was classified as Tetratrichomonas buttreyi according to its morphology as revealed by scanning electron microscopy. This flagellate was successfully maintained in a cysteine-peptone-liver medium. It is, to our knowledge, the first report of Tetratrichomonas buttreyi in Spain. This trichomonad appears to be a nonpathogenic commensal which often proliferates in fluid faeces.

Molecular detection of Rickettsia typhi in cats and fleas.

Background Rickettsia typhi 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.