Walter Basso

Bovine besnoitiosis emerging in Central-Eastern Europe, Hungary.

BackgroundBesnoitia besnoiti, the cause of bovine besnoitiosis, is a cyst-forming coccidian parasite that has recently been shown to be spreading in several Western and Southern European countries.FindingsClinical cases of bovine besnoitiosis were confirmed for the first time in Hungary, by histological, serological and PCR analyses.ConclusionsThis is the first report of autochthonous bovine besnoitiosis in Central-Eastern Europe. The emergence of bovine besnoitiosis in this region represents a further example, when human activity (i.e. cattle trading) is the main factor involved in the geographical spread of an infectious disease.

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

BackgroundToxoplasmosis 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 immunosupressed 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.MethodsIn 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.ResultsThe 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.ConclusionThe 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.

Bovine besnoitiosis in Switzerland: Imported cases and local transmission

Bovine besnoitiosis is an economically important disease of cattle, caused by Besnoitia besnoiti (Protozoa, Apicomplexa). A considerable spreading of this parasitic infection has been observed in Europe in the last ten years, mainly related to animal trade. In order to investigate the possibility of B. besnoiti being unnoticed introduced and getting established in Switzerland through the import of breeding cattle from France, a total of 767 animals (650 cattle imported from France and 117 cattle that had contact with B. besnoiti positive cattle in Swiss farms) were screened for antibodies against B. besnoiti by both a commercial ELISA and by the indirect fluorescent antibody test (IFAT). A total of 101 (13.17%) samples showed a positive reaction in ELISA (cut-off: percent of positivity [PP] ≥ 15) and 16 (2.09%) samples had IFAT titers ≥ 1:100. Eight of those samples reacted positive in Western blot (WB), corresponding to five imported Limousin cattle (two cows and one bull from France and two cows from Germany) and to three cattle born in Switzerland (one Limousin heifer born from one of the positive German cows, and two adult Braunvieh cows, that had been in contact with one of the French cows at a Swiss farm). Seven of those animals were subclinically infected and one animal showed only very mild signs. They were subsequently slaughtered, and the serological diagnosis could be confirmed by real-time PCR and/or histopathology in seven animals. The most frequent parasite localizations were the tendons and surrounding connective tissue of the distal limbs and the skin of the head region. Furthermore, B. besnoiti could be successfully isolated in vitro from one French, one German and one Swiss cattle (isolates Bb-IPZ-1-CH, Bb-IPZ-2-CH and Bb-IPZ-3-CH). In the current situation in Switzerland, prophylactic and control measures should include a serological examination of cattle to be imported from endemic areas and the culling of all confirmed positive animals from the herd. The evidence of B. besnoiti infection in both imported and locally born cattle shows that the conditions for the establishment and dissemination of this parasite in Switzerland seem to be adequate.

Novel tools for the diagnosis and differentiation of acute and chronic bovine besnoitiosis

Diagnosis of acute bovine besnoitiosis is a major diagnostic problem. We developed diagnostic tests to serologically diagnose and differentiate acute and chronic cases of bovine besnoitiosis using affinity purified antigens of Besnoitia besnoiti tachyzoites in immunoblots and in both, a conventional ELISA and an avidity ELISA. Sera of acutely and chronically infected cattle were investigated using these tests. Acutely infected cattle initially recognised an antigen of 74 kDa relative molecular mass, followed by reactions with increasing intensity against 81 and 28 kDa antigens. In addition, faint reactions against antigens with 36, 37, 39 and 42 kDa molecular mass started soon after seroconversion and increased over time. An antigen of 45 kDa molecular mass was transiently recognised early after infection but not or only weakly in the chronic stage. At least two antigens, the 39 and the 42 kDa antigens, seem to be located on the surface of B. besnoiti tachyzoites as determined by biotinylation. Affinity purified antigen was used to establish an APure-BbELISA which showed excellent sensitivity (100%) relative to a serological reference system in naturally, most likely chronically, infected cattle. Specificity was also high (99.8%) as determined in cattle from herds with Neospora caninum-associated abortions. The antibody levels in APure-BbELISA were correlated with the parasite load in the skin or the mucous membrane of the vestibulum vaginae as determined by real-time PCR. In acute cases of bovine besnoitiosis (confirmed by the detection of low avidity IgG in the APure-BbELISA) first specific antibodies were detected by ELISA in all animals except one, at the same time or earlier than in the serological reference system. The detection of parasite DNA in skin by real-time PCR was clearly superior to serological analysis in detecting infected cattle during acute besnoitiosis.

Epidemiological investigations of bovine besnoitiosis in Hungary

It usually gains proper attention, when blood-sucking arthropod species in Europe show northward spreading, in part due to climate change and global warming. Accordingly, it is well known, that the geographical regions where pathogens transmitted by these as biological vectors are endemic, also may extend towards the north. However, it is mostly less emphasized, that for vector-borne pathogens present in Western Europe the risk of eastward emergence is also possible, if they have competent vectors in the central and/or eastern part of our continent. Besnoitia besnoiti (Apicomplexa: Sarcocystidae) is a cyst-forming coccidian parasite that may cause severe lesions (with usually high seroprevalence, but low morbidity and mortality) in cattle as intermediate hosts. Wild ruminants are also susceptible. As a unique example among cystogenic coccidia, the main transmission route of B. besnoiti appears to be mechanical by blood-sucking dipterans (tabanid and stable flies), although it is also possible iatrogenically (with hypodermic needles) and most likely with close contact between animals. Bovine besnoitiosis has been endemic to South-Western Europe for more than a century, but a significant geographical expansion was observed during the last decade to other parts of the formerly endemic countries and to countries neighboring France. During the autumn of 2013 bovine besnoitiosis was diagnosed in a beef cattle herd in Hungary (for the first time in Central-Eastern Europe), following the import of Aubrac heifers and bulls from France in the previous two years. The preliminary serological herd screening with ELISA shows that even after the 2nd year of the presence of imported animals (with high prevalence of B. besnoiti infection), seropositivity among local animals, which originally belonged to the herd, is low. Based on ELISA results venereal transmission (from imported, infected bulls to local, uninfected cows) appears to be either rare or unlikely. The seroprevalence of besnoitiosis decreased significantly among calves born to the group of imported mother cows. The risk of infection seems to be high, when calves stay with their mother during suckling (for 6-7 months), and if animals are kept in the same stable (although physically separated) during the main fly season. Confirmation of the ELISA results is done with immunoblot and IFAT. All seropositive cattle are now kept at a distance of several kilometres from other groups of animals, prior to culling. Molecular and sero-epidemiological evaluation of the situation continues with the aim of preventing the spread of the disease and regaining the epidemiological status of Hungary as exempt of bovine besnoitiosis.

Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes

BackgroundThe apicomplexan parasite Toxoplasma gondii is cosmopolitan in nature, largely as a result of its highly flexible life cycle. Felids are its only definitive hosts and a wide range of mammals and birds serve as intermediate hosts. The latent bradyzoite stage is orally infectious in all warm-blooded vertebrates and establishes chronic, transmissible infections. When bradyzoites are ingested by felids, they transform into merozoites in enterocytes and expand asexually as part of their coccidian life cycle. In all other intermediate hosts, however, bradyzoites differentiate exclusively to tachyzoites, and disseminate extraintestinally to many cell types. Both merozoites and tachyzoites undergo rapid asexual population expansion, yet possess different effector fates with respect to the cells and tissues they develop in and the subsequent stages they differentiate into.ResultsTo determine whether merozoites utilize distinct suites of genes to attach, invade, and replicate within feline enterocytes, we performed comparative transcriptional profiling on purified tachyzoites and merozoites. We used high-throughput RNA-Seq to compare the merozoite and tachyzoite transcriptomes. 8323 genes were annotated with sequence reads across the two asexually replicating stages of the parasite life cycle. Metabolism was similar between the two replicating stages. However, significant stage-specific expression differences were measured, with 312 transcripts exclusive to merozoites versus 453 exclusive to tachyzoites. Genes coding for 177 predicted secreted proteins and 64 membrane- associated proteins were annotated as merozoite-specific. The vast majority of known dense-granule (GRA), microneme (MIC), and rhoptry (ROP) genes were not expressed in merozoites. In contrast, a large set of surface proteins (SRS) was expressed exclusively in merozoites.ConclusionsThe distinct expression profiles of merozoites and tachyzoites reveal significant additional complexity within the T. gondii life cycle, demonstrating that merozoites are distinct asexual dividing stages which are uniquely adapted to their niche and biological purpose.

First cases of besnoitiosis in cattle in Switzerland

Bovine besnoitiosis has been diagnosed in neighboring countries but not in Switzerland so far. This disease occurs endemically in France and focal outbreaks have been reported in Germany and Italy. To determine if Besnoitia besnoiti is introduced into Switzerland through the import of breeding cattle from France, a systematic serological survey was performed. A total of 412 breeding cattle (from 114 farms) imported from France into Switzerland between 2005 and 2011, were serologically examined for antibodies against B. besnoiti using a commercial ELISA kit (PrioCHECK© Besnoitia Ab 2.0, Prionics AG, Zurich, Switzerland). Sixty-four (15.5 %) animals reacted positive in ELISA. The serologic diagnosis was confirmed by an indirect immunfluorescence test (IFAT) and a Western blot (WB) in only 2 Limousin cows imported from France on a farm in Eastern Switzerland. Subsequently, this whole herd (n = 16) was examined clinically and serologically and 2 additional Limousin cows imported from Germany also reacted positive in the three serological tests. One of these cows presented B. besnoiti tissue cysts in the scleral conjunctiva and typical skin lesions in the head region. The infection was further confirmed cytologically, histopathologically and by PCR. It can be concluded that the parasite is most likely being introduced into Switzerland through the import of infected animals.

Toxoplasma gondii infection in sentinel and free-range chickens from Argentina

This study aimed at isolating and genotyping Toxoplasma gondii from serologically positive free-range chickens from Argentina, and to evaluate the use of sentinel animals during a short time period of exposure to determine environmental contamination with T. gondii oocysts. Two groups of chickens on six farms were compared in this study: (i) young, 2-3 month-old broiler-type chickens reared as sentinel animals on the farms and (ii) adult chickens reared on the same farms for more than one year. Seroconversion rates of 7.0% or 5.7% were observed in sentinel broiler chickens reared for a period of 74 days (January-April 2010) or 88 days (August-November 2010) respectively, as shown by a T. gondii specific immunofluorescent antibody test. Fifty-three percent (17 of 32) of adult chickens were positive and showed higher titres than sentinel animals. Isolation of T. gondii from tissues (brain and heart) of serologically positive chickens was achieved from six of seven free-range adult birds with IFAT titres of 200 and higher. The isolated parasites were analysed by multi-locus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The isolated T. gondii showed three different genotypes: two genotypes consisted in atypical allele combinations, and the remaining genotype had exclusively clonal type II alleles. All isolates obtained at a single farm, corresponded to the same genotype. The T. gondii genotypes observed are identical to those described in cats, dogs, chickens and capybaras elsewhere in South America. Two isolates, which showed different allele combinations in PCR-RFLP, were characterized in a mouse virulence assay. While one isolate showed a low virulence a second isolate was of intermediate virulence to mice.

Capillaria plica (syn. Pearsonema plica) infection in a dog with chronic pollakiuria: Challenges in the diagnosis and treatment

Capillaria plica (syn. Pearsonema plica) is a nematode parasite of the urinary tract of canids, felids and mustelids, which can cause cystitis, pollakiuria, dysuria and hematuria. An eight-month-old female crossbred dog from Switzerland presented a six-month history of frequent urination. During the first clinical examination, C. plica eggs were detected in the urine sediment. Three series of treatments with fenbendazole (50 mg/kg body weight[BW]/day, orally) for 10 days each, three single day treatments with moxidectin-imidacloprid (spot-on) and one single administration of ivermectin (0.2 mg/kg BW subcutaneously) were performed within an eight-month period. None of those treatments succeeded in eliminating the C. plica infection or in resolving the clinical signs. An endoscopic examination of the urine bladder still revealed numerous adult viable C. plica worms attached to the bladder mucosa. A two-day treatment with levamisole (7.5 mg/kg BW/day intramuscularly) was subsequently performed. An endoscopic control of the urine bladder two days after this treatment and a urine analysis after two weeks confirmed the elimination of the parasites. The clinical signs disappeared within one month. Levamisole was shown to be effective against C. plica infection in a dog, whereas previous treatments with fenbendazole, moxidectin and ivermectin had failed.

Efficacy of Emodepside/Praziquantel Spot-on (Profender®) against adult Aelurostrongylus abstrusus Nematodes in Experimentally Infected Cats

The adulticidal efficacy of a topical combination of emodepside 2.1 % (w/v) plus praziquantel 8.6 % (w/v) (Profender® spot-on for cats, Bayer) against adult Aelurostrongylus abstrusus nematodes was evaluated in two randomised, placebo-controlled laboratory efficacy studies. Each study involved 16 cats experimentally inoculated with L3 (800 and 600 each in studies no. 1 and 2, respectively) and randomised into two study groups of 8 cats each after onset of patency. While cats in the treatment group in study no. 1 received a single spot-on application at the minimum therapeutic dose (3 mg/kg emodepside and 12 mg/kg praziquantel), cats in study no. 2 were treated twice with an interval of 14 days. The faecal output of first stage larvae was monitored throughout the study. Necropsy was conducted 4 or 5 weeks after the (first) treatment and the worm counts were used for efficacy calculations. The control groups showed a geometric mean of the total worm count (live and dead worms) of 28.8 (study no. 1) and 17.6 (study no. 2), respectively. All control animals were infected. While the single treatment in study no. 1 resulted in a reduction of the total worm burden by 73.0 % (p = 0.0070), the treatment protocol in study no. 2 was 99.2 % effective (p = 0.0035). Based on live worm counts, the efficacy in study no. 2 was 100 % (p = 0.0030). It is concluded that two applications of Profender® spot-on given two weeks apart represent a safe and highly efficacious treatment regime against feline aelurostrongylosis.