Risk assessment of transfusion-associated babesiosis in Tyrol: appraisal by seroepidemiology and polymerase chain reaction.

BACKGROUND: After malaria, babesiosis is the second most common transfusion-transmitted parasitic disease in the United States. In Europe, one reported transfusion case, concerning Babesia microti, occurred in Germany. STUDY DESIGN AND METHODS: Due to the fact that Babesia spp. are present in Tyrolean ticks, the aim of this study is to assess the occurrence of immunoglobulin (Ig)G antibodies against the Babesia divergens complex, including B. divergens and Babesia venatorum (EU1), as well as B. microti by screening a representative collective of 988 blood donors from North and East Tyrol (Austria) with indirect immunofluorescence antibody test. Additionally, we investigated 206 local ixodid ticks for the presence of babesial DNA by polymerase chain reaction. RESULTS: Seroprevalence data resulted in rates of 2.1% for IgG antibodies against the B. divergens complex and 0.6% against B. microti in Tyrolean blood donors. All sera could be confirmed by independent retesting. Our data indicate that cross-reactivity is high between B. divergens and B. venatorum and lower than 19.8% between B. divergens and B. microti. CONCLUSIONS: This study shows that Babesia spp. are present in the Tyrols, which blood donors come into serologic contact with, and that we have to consider how to sustain blood product safety concerning this new challenge. Additionally, it is the first description of B. venatorum in the Tyrols, found in one Ixodes ricinus at the Italian border.

Evaluation of a commercial ELISA kit for detection of antibodies against Toxoplasma gondii in serum, plasma and meat juice from experimentally and naturally infected sheep.

BACKGROUND: Toxoplasmosis is one of the most common food borne zoonoses worldwide, and can be a serious life-threatening disease in the congenitally infected fetus and in immunosuppressed patients. Among food animals, sheep along with goats and pigs possess the highest incidence of T. gondii cysts in meat, and play a major role as a source of human infection. METHODS: In this study, a new commercial ELISA kit (PrioCHECK Toxoplasma Ab SR, Prionics Schlieren-Zurich, Switzerland) for the detection of anti-T. gondii antibodies in serum, plasma and meat juice of sheep, was evaluated by comparing it with the indirect fluorescent antibody test (IFAT), indirect haemagglutination test (IHA) and real-time PCR, on samples from experimentally inoculated and naturally exposed sheep. RESULTS: The commercial ELISA detected the infection status in 50% and 100% of sheep orally inoculated with 10,000 T. gondii oocysts (n = 6), from two or three weeks post infection (wpi), respectively, both on serum and plasma samples. Meat juice from all experimentally inoculated sheep collected at slaughter (12 wpi) showed positive ELISA values. In naturally exposed sheep (n = 396), the ELISA showed a very good agreement with IFAT (kappa = 0.91-1.0) and IHA (kappa = 0.96-1.0) performed on serum; and a positive correlation was observed between ELISA values and IFAT titers. By a Receiver Operating Characteristics (ROC) curve analysis, the commercial ELISA had relative sensitivities between 93.33% and 100%, and relative specificities between 96.87% and 100% respect to IFAT and IHA, depending on the considered cut-off value and animal groups tested. Furthermore, the ELISA correctly recognized all animals reacting positive in real-time PCR. The ELISA results on meat juice agreed with those on serum samples in all experimentally inoculated animals, and in 94 out of 96 (97.9%) naturally exposed sheep, when meat juice was tested at a 1:10 dilution. CONCLUSION: The commercial ELISA kit evaluated in this study could represent a valuable tool to improve the surveillance and reporting system for T. gondii in sheep populations at the farm level or for diagnosis at the slaughterhouse, contributing to the control of this widespread zoonosis.

[Vector-borne parasites of dogs on the Islands of Cabo Verde]. / Untersuchungen zu vektorübertragenen Parasiten bei Hunden auf den Kapverdischen Inseln.

During an animal welfare campaign on the Islands of Cape Verde (in the capital Praia on the island of Santiago) a total of 57 dogs were examined for ectoparasites and blood parasites. 84% of the animals were infested with arthropods, mostly ticks. Haemotropic parasites were examined via blood smear, polymerase chain reaction (PCR) and/or serology. 23 dogs had single infections, five multiple infections with haemoparasites. In eight dogs Ehrlichia canis and in one Amaplasma phagocytophilum were detected by PCR, while 43.1% and 36.2% respectively were serologically positive. In 23 blood smears Babesia gibsoni, but no Babesia canis could be detected. In four cases Hepatozoon canis was found in the smears. All animals were negative for Dirofilaria larvae or antibodies against Leishmania. Arthropod-infested animals more frequently harboured babesiae (44%), ehrlichiae (19%) or H. canis (8%) compared to non-infested animals (20%, 0%, 0%). In bitches and dogs aged one year or less babesiae were more frequently detected (48% of the bitches and 13% of the male dogs; 45% of animals less than one year and 26% of the older ones). Due to the high infection rates the import of animals from Cabo Verde to Central Europe must be seen as critical, since an endemisation of (sub-)tropical infectious agents cannot be excluded.

[Arthropod-borne parasites of dogs, especially Leishmania, in the Kosovo and Albania]. / Infektionen mit arthropodenübertragenen Parasiten bei Hunden im Kosovo und in Albanien unter besonderer Berücksichtigung der Leishmanieninfektionen.

Currently no information is available regarding canine arthropod-borne parasites in Albania and the Kosovo, especially the zoonotic protozoan Leishmania infantum. Presumably autochtonous cases of human leishmaniosis have been described for some areas (Kosovo: Ferizaj, Gjakovo, Pec, Malisevo; Albania: Tirana, Durres, Elbasan, Shkodra, Vlore). In order to investigate the infection status of dogs of different origin sera from 272 animals (151 from Albania - Tirana, Kamza and Durres; 121 from the Kosovo - Gjakovo, Ferizaj and Prishtina) were obtained. Corresponding blood samples were available from 36 Albanian stray dogs. Antibody titres were determined by Indirect Immunofluorescence Test against L. infantum and Babesia canis. Antigens of Dirofilaria immitis were determined using the DiroCheck-Test. Blood samples were tested for L. infantum, B. canis, Hepatozoon canis, D. immitis and Dirofilaria repens by polymerase chain reaction (PCR). Specific antibodies against Babesia were found in 7.3% of the animals, against Leishmania in 3.3% and 7.0% were positive for D. immitis antigen. DNA of Babesia, Leishmania or D. repens was detected in 11.1%, respectively. H. canis was found in 52.8% of the blood samples. D. immitis DNA was not detected. Since the Babesia- and Leishmania-PCR-positive animals were all serologically negative it can be assumed that infections were acquired only recently. All Leishmania-positive animals were stray dogs. These animals contribute to the maintenance of Leishmania transmission in endemic areas, and a control of the canine stray population should be considered.

Babesia divergens-like organisms from free-ranging chamois (Rupicapra r. rupicapra) and roe deer (Capreolus c. capreolus) are distinct from B. divergens of cattle origin - an epidemiological and molecular genetic investigation.

In 2005 and 2006, three adult female chamois (Rupicapra r. rupicapra) were found dead with signs of acute babesial infection in the eastern Swiss Alps. PCR on DNA extracted from blood or spleen of the carcasses revealed sequence identity of the amplified part of the 18S rRNA gene with GenBank entries attributed to Babesia divergens of cattle origin or B. capreoli of wild ruminant origin which have never been described before in this region. Examination of 424 blood samples from 314 head of cattle from this area by IFAT, microscopy and PCR provided no evidence for babesial infection. Six of 887 ticks collected from cattle were PCR-positive, and sequencing revealed Babesia sp. genotype EU1 in five and B. divergens/B. capreoli in one of them. A Babesia isolate of chamois, two isolates of roe deer from the same region and one isolate of a roe deer from the north-western Swiss Alps were genetically compared with two Swiss B. divergens isolates of cattle origin by analysing the genomic rDNA locus. Whereas the near full length sequences of the 18S rRNA gene were virtually identical among all six isolates (>99.4% identity), distinct differences between the two isolates from cattle on the one hand and the four isolates from free-ranging ruminants on the other hand were observed in the sequences of the internal transcribed spacers 1 and 2 (ITS1, ITS2) and part of the 28S rRNA gene. These results indicate that, albeit genetically very closely related, these babesial organisms from cattle and from free-ranging ruminants indeed are distinguishable organisms with different host specificities, and they support the use of the discrete species name B. capreoli for the B. divergens-like organisms from chamois and roe deer.

Seroprevalence of Toxoplasma gondii and Neospora caninum infection of cats in Hungary.

Blood samples were collected from 330 cats in Hungary in order to evaluate their seroconversion to Toxoplasma gondii and Neospora caninum using the indirect fluorescent antibody test (IFAT). The overall prevalence of toxoplasmosis was 47.6%, the prevalence being 22.4% among urban, 50% among suburban and 61.3% among rural animals. Significantly more cats had high IFAT titres (1:640 to 1:5120) in the countryside. Female cats were more frequently infected with T. gondii than males (53.3% vs. 39.3%), and seropositivity increased with the age of animals. The prevalence (0.6%) and titre (1:40) of antibodies to N. caninum was low. Sixty-two cats were also screened for seroconversion to feline infectious peritonitis (FIP) virus. Higher titres to T. gondii were more frequently detected among FIP-positive cats, but this difference was non-significant due to the small number of cats with concurrent infection.

Clinical symptoms and diagnosis of encephalitozoonosis in pet rabbits.

Infections with Encephalitozoon cuniculi in rabbits are observed at increasing frequency and are known as opportunistic infections in immunocompromised humans. 191 pet rabbits with suspected encephalitozoonosis, presented at the Animal Hospital of the Veterinary University of Vienna (Austria), were included in this study. Rabbits were serologically examined for antibodies against E. cuniculi (144 positive out of 184 rabbits with suspected encephalitozoonosis compared to 14 positive out of 40 clinically healthy rabbits tested as part of a standard health check) and Toxoplasma gondii (8 positive out of 157). Of the 144 seropositive rabbits with clinical signs, 75% showed neurological symptoms, 14.6% demonstrated phacoclastic uveitis and 3.5% suffered from renal failure. 6.9% of the animals had combined symptoms. Vestibular disease dominated within the rabbits that showed neurological symptoms. Polymerase chain reaction (PCR) could not detect parasite DNA in urine or cerebrospinal fluid (CSF), but did so in 4 out of 5 samples of liquefied lens material in cases with phacoclastic uveitis due to lens capsule rupture. Additionally further diagnostic procedures, such as inspection of the external ear canal (N=69), radiography of the tympanic bullae (N=65) were performed to rule out differential diagnosis. 54.2% of the patients exhibiting neurological symptoms recovered within a few days, while 87.5% of the rabbits suffering from renal failure died or had to be euthanized.

Imported non-endemic, arthropod-borne and parasitic infectious diseases in Austrian dogs.

BACKGROUND/AIMS: An increasing number of imported non-endemic arthropod-borne diseases and other rare parasitic diseases in dogs in Austria were the basics for this report. Number and species of imported pathogens as well as the origin of the carrying dogs were of special interest. METHODS: Symptomatic dogs that have been in a foreign country before were included to the study. 174 dogs, presented at the Veterinary University of Vienna, were analysed for their origin or stay in a foreign country (n = 26) and type of infection. The age and breed distribution was recorded. Dogs were tested for leishmaniosis, ehrlichiosis, filariosis and infection by Rickettsia conorii. RESULTS: Overall infection rate was 47% in the tested dogs. Mixed infections were diagnosed in 10% of dogs included. Infestation by Cordylobia anthropophaga and Pentastomida was documented. CONCLUSIONS: The introduction of non-endemic pathogens, and sometimes their vectors, by dogs is documented in Austrian dogs. Direct transmission from dogs to humans is very unlikely and most of the vectors (Phlebotomus spp.) are not native in Austria. A future risk may arise from an increasing number of imported dogs, carrying these vectors that may be host to various pathogens, to areas still free of those pathogens. A further problem is the probability, that these vectors may become native when climate conditions are going to be favourable to them.

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.

[Tick infestation and the prevalence of Borrelia burgdorferi and Babesia divergens in cattle in Bavaria]. / Untersuchungen zum Zeckenbefall sowie zur Prävalenz von Borrelia burgdorferi und Babesia divergens bei Rindern in Bayern.

During the grazing period 2002 319 cattle from 31 farms located in 6 districts of southern Bavaria were examined for the presence of ticks in 4- to 5-week intervals, and 287 serum samples were tested for the presence of antibodies against Borrelia burgdorferi and Babesia divergens. Ticks were detected in all 31 farms with a mean prevalence of 69%. 3218 out of 3453 collected ticks were Ixodes ricinus; 139 nymphs, 19 larvae and 77 damaged adult specimens could only be determined to the Genus level (Ixodes). The seasonal pattern revealed the highest frequencies of ticks in May/June and September. The intensity of tick infestation of positive animals was generally low. 76.5% of parasitized cattle had 1-6 ticks per day of investigation. Individual cattle showed up to 250 ticks per day. The percentage of infested animals in each herd varied within the period between 0-100%. The examination of serum samples by immunofluorescence technique (IFAT) revealed positive anti-Borrelia antibody titers (> or = 1:64) for 45.6% of the animals. The within-farm seroprevalence of borreliosis ranged from 20 to 100% in 27 of the 31 farms. A significant correlation could be detected between the number of ticks/cattle and the anti-Borrelia burgdorferi IgG-titer. By contrast, there was no significant correlation between the age of the animals and anti-Borrelia serum titers. For comparative reasons, 64 IFAT-positive serum samples were tested by Western blot techniques for the presence of antibodies cross-reacting with Borrelia garinii antigen. These analyses revealed that 69% of the samples reacted positively, 28% were unclear and 3% were negative. Examinations of the 287 serum samples for the presence of anti-Babesia divergens antibodies revealed one positive animal with a titer of 1:16.

Babesia divergens becoming extinct in cattle of Northeast Hungary: new data on the past and present situation.

Previously unpublished data from 1958 to 1967 attest the occurrence of Babesia divergens in cattle in several endemic foci of Northeast Hungary. During that period the number of clinical cases showed fluctuation with intervals of 4-5 years and monophasic seasonality (peaking in June). In order to assess the current status of bovine babesiosis in that region, blood samples were collected from 654 cattle on 44 farms of 36 settlements in or near the endemic area during 2005, and serum levels of IgG antibodies to B. divergens were measured by indirect fluorescent antibody test (IFAT). Only 2 samples (0.3%) showed positivity. In one village clinical babesiosis was observed over the past few years. Animals brought into the endemic area during the spring developed haemoglobinuria in the summer of the same year, but those introduced during the summer or autumn showed clinical signs only after two years. Sampled animals born and raised locally had neither haemoglobinuria nor seroconversion. Reduction in the number of cases during the past decades may have been influenced by the availability of hosts (i.e. decrease of cattle breeding) and the activity of vectors associated with climate-related changes (e.g. increase of annual sunlight hours in the endemic area). This is the first report on the prevalence of antibodies to B. divergens in cattle in Hungary.

Seroprevalence of neosporosis in beef and dairy cattle breeds in Northeast Hungary.

In order to assess the seroprevalence of bovine neosporosis with indirect fluorescent antibody test (IFAT), blood samples were collected randomly from 1063 beef and dairy cattle belonging to 12 different breeds in Northeast Hungary. Antibodies to Neospora caninum were detected in 27 (2.5%) of the animals, kept on 19 of the 42 settlements included in this survey. Since samples were collected on 50 farms, herd prevalence amounted to 38%. The percentage of cattle with seroconversion increased with age, suggesting a postnatal source of infection. The highest rate of positivity was detected in Aberdeen Angus (3.3%) and Holstein-Friesian cows (3.2%), and the lowest in Limousine (0.9%), but no breed predisposition was statistically substantiated. Neosporosis was more prevalent in dairy (3.4%) than in beef (1.9%) cattle, although the difference was not significant. Only three out of the seropositive cows, all of them Holstein-Friesians, had a history of abortion.

Differentiation of Babesia bigemina, B. bovis, B. divergens and B. major by Western blotting--first report of B. bovis in Austrian cattle.

To establish an assay for the serological differentiation of bovine Babesia species ( B. bigemina, B. bovis, B. divergens and B. major), antigens from experimentally infected cattle were Western blotted and probed with homologous and heterologous sera. Varying antigen patterns for each species allowed the determination of species-specific diagnostic antigens. Blood samples from 36 naturally infected cattle from the province of Styria were tested by indirect immunofluorescence antibody test (IFAT) against B. divergens, as well as by Western blotting against B. bigemina, B. bovis, B. divergens and B. major, 3 weeks after clinical babesiosis was diagnosed by blood smears. All 36 cattle were B. divergens-positive when tested by IFAT. In four cases (11%), an infection with both B. bovis and B. divergens and in two cases a single infection with B. bovis were diagnosed when tested by Western blot. B. bigemina and B. major infections were not detected. These are the first serologically confirmed cases of B. bovis in Austrian cattle.

Molecular characterization of a non-Babesia divergens organism causing zoonotic babesiosis in Europe.

In Europe, most reported human cases of babesiosis have been attributed, without strong molecular evidence, to infection with the bovine parasite Babesia divergens. We investigated the first known human cases of babesiosis in Italy and Austria, which occurred in two asplenic men. The complete 18S ribosomal RNA (18S rRNA) gene was amplified from specimens of their whole blood by polymerase chain reaction (PCR). With phylogenetic analysis, we compared the DNA sequences of the PCR products with those for other Babesia spp. The DNA sequences were identical for the organism from the two patients. In phylogenetic analysis, the organism clusters with B. odocoilei, a parasite of white-tailed deer; these two organisms form a sister group with B. divergens. This evidence indicates the patients were not infected with B. divergens but with an organism with previously unreported molecular characteristics for the 18S rRNA gene.