Prevalence and analysis of potential risk factors for Cryptosporidium parvum infection in lambs in Zaragoza (northeastern Spain)

An epidemiologic study was carried out to investigate the prevalence of and to identify factors associated with the risk of Cryptosporidium infection in sheep in Zaragoza (northeastern Spain). Faecal samples from 583 lambs aged from 1 day to 3 months and 205 ewes older than 1 year were collected at 89 farms in the two regions of the province of Zaragoza with the highest sheep population (Zaragoza and Ejea de los Caballeros). In every sheep farm, data of the factors potentially associated with the likelihood of C. parvum infection were analysed: geographical location, season, size of herd, number of lambs in the herd at sampling time, lambing period, cleaning of lambing area and presence of diarrhoeic lambs in the farm. C. parvum oocysts were identified by using the Ziehl-Neelsen technique in 344 lambs (59%) from 75 farms (84.4%). Infected lambs ranged from less than 7 days to 90 days of age, although the percentage of animals shedding oocysts peaked at 8-14 days of age (76.2%). Statistical analysis showed that infection rates were significantly higher in lambs aged between 1 and 21 days (66.4%) than in those aged between 22 and 90 days (23%) (P<0.0001, chi(2)). Analysis of correlation between excretion of oocysts and diarrhoea revealed a relationship in all age groups and the probability of presenting diarrhoea was significantly higher for lambs shedding oocysts (86.3%) than for those which did not excrete the parasite (32.2%) (P<0.0001, chi(2)). Similarly, cryptosporidial infection rates were significantly higher in diarrhoeic (79.4%) than in non-diarrhoeic lambs (22.4%). Furthermore, infection intensity was correlated with the presence of clinical symptoms. Presence of diarrhoeic lambs in the farm was the only factor significantly associated with an increased risk of infection since the percentage of herds testing positive was significantly higher in farms with diarrhoeic lambs (91.3%) than in those without cases of neonatal diarrhoea (12.5%) (P<0.0001, chi(2)). Factors associated with a decreased risk of C. parvum infection in lambs included low numbers of lambs in the farm and cleaning of the lambing area. Additionally, lambs 8-14 days of age were less likely to be infected at the first lambing period and in spring/autumn. Cryptosporidial infection was also detected in 16 ewes (7.8%) which excreted few oocysts and without diarrhoea.     

Molecular phylogenetic analysis of Onchocerca lupi and its Wolbachia endosymbiont

The morphology of Onchocerca lupi, responsible for canine ocular onchocercosis, is unique within the genus. Earlier analyses of the 5S ribosomal RNA gene spacer region sequence of the parasite and the 16S ribosomal RNA gene sequence of its Wolbachia endosymbiotic bacteria (Rickettsiales) supported the morphological and biological arguments that O. lupi is a distinct species. However, the exact phylogenetic position of O. lupi and its endosymbiont could not be unambiguously determined. Herein we report analyses based on the mitochondrial cytochrome oxidase I (COI) gene of the filarial species and the Wolbachia surface protein (wsp) and the bacterial cell-cycle ftsZ genes of their wolbachiae. Our results indicate that O. lupi separated from other Onchocerca spp. early in evolution. This is in line with the previous morphological analysis demonstrating that O. lupi is an atypical Onchocerca species showing both primitive and evolved characters. The phylogenetic trees generated for the COI sequences of filariae and the wsp and ftsZ sequences of their wolbachiae were congruent with each other, which supports the hypothesis that nematodes and their Wolbachia endobacteria share a long co-evolutionary history.     

Serological evidence for Babesia canis infection of horses and an endemic focus of B. caballi in Hungary

In order to evaluate the seroconversion of horses to Babesia caballi and B. canis in Hungary, blood samples were collected from 371 animals on 23 different locations of the country. The presence of antibodies to B. caballi was screened with a competitive ELISA. All 29 positive samples came from one region (the Hortobágy). The prevalence of infection did not show correlation with sexes, and reached 100% in the age group of 2-5 years. Babesia canis-specific antibodies were demonstrated by IFAT in 6.74% of animals kept in 7 regions. The titres were low or medium level (1:40 to 1:160), indicating that the horses had previously been exposed to this piroplasm, but their infection must have been limited. The highest seropositivity rate was observed in the age group of 3-4 years, and males (stallions and geldings) were significantly more frequently infected than females. However, neither B. caballi nor B. canis could be identified in the peripheral blood samples of infected horses by PCR. Since most of the B. caballi-positive horses remained negative in the B. canis IFAT, whereas seroconversion solely to B. canis was detected in several regions of the country, serological cross-reaction between the two species can be discounted. This is the first serological evidence of horses being naturally infected with B. canis, supporting the view that piroplasms are less host specific than previously thought.     

Study on the inductive factors of hypobiosis of Ostertagia ostertagi in cattle

Two experiments were carried out to determine the causes producing the Ostertagia ostertagi hypobiosis phenomenon in cattle. In the first experiment, the effect of time on third-stage larvae in the environment was studied during a 2-year period. Three experimental paddocks contaminated with O. ostertagi eggs at different times of the year were used, and the levels of hypobiosis were recorded by using 'indicator' and 'tracer' calves. The results suggest that time as such is not a hypobiosis-inductive factor. The second experiment was conducted under laboratory conditions, where the effects of temperature and light on infective larvae were studied. Infective larvae were subjected to different conditions of temperature and light during 6 weeks, and then inoculated to parasite-naive calves, which were slaughtered after 4 weeks. Percentages of hypobiotic larvae in these calves varied from 3.5 to 94.8%, depending on the different storage conditions the larvae underwent before inoculation. Results suggest that increasing temperature and increasing time of light exposure simulating spring conditions would be the factors which act upon third-stage larvae inducing them to a later hypobiotic stage in the host.     

Model field study of Sumithion 50 EC and Fusilade S on pheasants.

Toxicological studies on wild animals play an important role in the ecotoxicological examination of pesticides. The applied model tests enable the assessment of toxicological consequences with particular regard to the life and nutrition of wild animals in the ploughed field among plants treated with pesticides. The application of different pesticide formulations on plough-land may pose a simultaneous chemical burden to wild birds. In this model study, manifestations of the interaction between an insecticide and a herbicide were studied in pheasants. The birds were placed on lucerne in cages (48 m2) and sprayed once. The applied doses were: Sumithion 50 EC 1 litre/ha + Fusilade S 6 litres/ha (practical doses) and Sumithion 50 EC 5 litres/ha + Fusilade S 30 litres/ha. The analytically determined pesticide concentration of the lucerne was taken as a basis in the further treatment of fodder. The fodder of pheasants contained the following chemicals: 85 mg/kg Sumithion 50 EC + 510 mg/kg Fusilade S and 425 mg/kg Sumithion 50 EC + 2250 mg/kg Fusilade S. Sporadic deaths observed among the pheasants were of traumatic origin and not due to a toxic effect. The decrease of body weight was significant only at the higher dose levels. Acetylcholinesterase (AChE) activity of the blood decreased significantly in both dose groups. On the basis of the results obtained it can be established that at the dose level used in the practice the pesticides studied do not give rise to a toxic interaction in pheasants.     

Ochratoxin a content of urine samples of healthy humans in Hungary

The ochratoxin A (OTA) content of urine samples from 88 healthy humans living at five settlements in three counties of Hungary was determined by immunoaffinity column cleanup and high-performance liquid chromatography (HPLC). OTA was detected in 61% of the samples in an average concentration of 0.013 ng/ml (range: 0.006-0.065 ng/ml). OTA concentrations measured in urine samples from men and women were not significantly different. The OTA concentration of samples from Heves county was significantly (t-test; p < 0.003) higher than that of samples from Hajdú-Bihar and Somogy counties. The regional differences in OTA concentration of urine samples indicate regional differences in the OTA exposure of the human population. Further studies are necessary to determine the cause of the regional differences in the OTA intake. The studies allow us to conclude that the OTA intake of the majority of the Hungarian population is low (< 1 ng/kg of body weight per day) but a certain part of the rural population may take up higher levels of OTA.     

Biological and DNA evidence of two dissimilar populations of the Rhipicephalus sanguineus tick group (Acari: Ixodidae) in South America

In this work, the biology, mitochondrial DNA and fertility of hybrids from two strains of Rhipicephalus sanguineus, from Brazil and Argentina, were compared. Engorged larvae, nymphs and adults from Argentina weighed more and the engorgement period of adult females was significantly longer than those of their Brazilian counterparts, whereas adult female tick yield rate was higher for the Brazilian strain. High intraspecific divergence of mitochondrial DNA was detected between R. sanguineus from Brazil and Argentina. On the other hand, a strong genetic relationship was detected between European and Argentinean R. sanguineus populations while the Brazilian population appeared to be related to the African Rhipicephalus turanicus. Adult hybrid females laid eggs, which were mostly unviable, whereas a mean of more than 1400 larvae hatched per egg mass from pure Brazilian and Argentinean strains. These results showed that differences between these strains are greater than previously assumed and that the biosystematic status of R. sanguineus ticks from South America should be re-evaluated. Wide variations, such as these might account for the reported worldwide differences in biology and vector capacity of this species.     

Detection of bovine herpesvirus 4 in CD11b+ leukocytes of experimentally infected rabbits

The presence and numbers of bovine herpesvirus 4 (BoHV-4) infected CD11b+ leukocytes were investigated during experimental infections of New Zealand White rabbits by Fluorescence Activated Cell Sorter (FACS) analysis. Peripheral blood leukocytes (PBL) were collected every second day, and the cells were stained with phycoerythrin-labelled CD11b-specific mouse monoclonal antibody and fluorescein-conjugated bovine herpesvirus 4-specific mouse monoclonal antibody. The numbers of double-stained cells from PBLs of the control and inoculated groups were measured and compared in FACSTREK analyser. Double-stained cells were detected in the virus-inoculated group on postinoculation days (PID) 2-5 and 9-12. The results indicated that CD11b+ PBLs were permissive for BoHV-4 infection, and are probably the main reservoir of the virus during the latent period. The data did not indicate production of infectious viral particles, but virus-specific proteins were expressed on the surface of CD11b+ cells. The two waves of double-stained cells gave similar results to the PCR assays from serum samples, which showed the presence of viral DNA in the serum on the same days when virus-infected CD11b cells were also present. Productive BoHV-4 infection of mast cells or undifferentiated leukocytes in the bone marrow and the antiviral immune response might be responsible for this periodic appearance of the virus in CD11b+ PBLs and in the serum. The paper provides evidence that CD11b+ PBLs are the main target cell populations in the blood for BoHV-4.