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Serological cross-reactions represent a serious problem in some currently available tests to diagnose Besnoitia infections in many species including cattle, caribou and donkeys. False-positive results are due to the low positive-predictive value of these serological tests for besnoitiosis. These tests therefore have clear limitations if large herds are screened in areas with low prevalence, since increased numbers of false-positive reactions require confirmatory testing by alternative serological methods, e.g. immunoblotting, which are time-consuming and create extra costs. To overcome this problem, we aimed to develop a highly sensitive and specific competitive ELISA (cELISA) using a panel of 12 monoclonal antibodies raised against the tachyzoite stage of Besnoitia besnoiti. A cELISA set up with one of these antibodies (Bb-cELISA1) was screened with a large panel of B. besnoiti-positive bovine sera to estimate the diagnostic sensitivity of the test. Sera from herds with Neospora caninum- or Sarcocystis spp.-infected cattle were used to estimate its diagnostic specificity. Relative to a reference standard, which combined the results obtained in a previously established highly sensitive and specific ELISA, in the immunofluorescence antibody test and in B. besnoiti tachyzoite and bradyzoite immunoblots, the new Bb-cELISA1 revealed a diagnostic sensitivity of 99.2% (95% confidence interval: 97.1-99.9%) and a diagnostic specificity of 99.9% (95% confidence interval: 97.7-100%). This novel assay was tested on a variety of proven Besnoitia-positive sera from other species, including B. besnoiti-infected cats, rabbits or Besnoitia bennetti-infected donkeys or Besnoitia tarandi-infected caribou. The results obtained with the new Besnoitia-cELISA for these animal species also corresponded almost perfectly with those of the reference tests, which included immunoblot and immunofluorescence antibody tests. In conclusion, the novel Besnoitia-cELISA represents a valuable tool for the diagnosis and control of bovine besnoitiosis and for studies on the epidemiology of Besnoitia infections in a variety of host species, including naturally exposed wildlife and experimental hosts., (Copyright © 2020 Australian Society for Parasitology. All rights reserved.) more...

Background: Bovine besnoitiosis has been recently diagnosed in a three-parted herd of 796 Aubrac and Charolais beef cattle in Hungary. A large scale serological, histological and molecular survey was initiated in order to uncover important factors in the local epidemiology of the disease., Findings: Blood samples were collected (three times from the whole herd, and repeatedly from selected animals) for serological screening by ELISA. In addition, various organs from aborted fetuses and newborn calves, skin and colostrum samples of seropositive heifers/cows, and ticks collected from the cattle were histologically and/or molecularly analysed for the presence of Besnoitia besnoiti. All fetal and calf tissues, as well as colostrum and tick samples from cows were PCR negative. Based on ELISA results, only very few local cows seroconverted after mating with imported, infected bulls, and not necessarily as a consequence of this event. Among calves that were born to seropositive, imported cows and stayed with their mother until weaning at seven months of age, seroprevalence decreased significantly, but remained high. At the same time, 28 calves born from seropositive cows, but separated from their dams immediately after receiving colostrum, were successfully reared and remained uninfected. Following a second herd-level screening, all Aubrac cattle (except for heifer calves) and all seropositive Charolais cows and bulls were culled. Manifestation of the disease is currently sporadic. Among chronically affected heifers two types of skin lesions were noted, and histological evaluation indicated marked distension of sweat gland ducts with membrane-bound structures (resembling cystozoites) in their contents., Conclusions: Transmission through natural mating, as well as transplacental, colostral and tick-borne transmission of B. besnoiti was either unlikely or did not occur. However, the risk for spreading of the infection was high, when calves stayed with their mother during suckling, and if animals were kept in the same stable (although physically separated) during the main fly season. Herd replacement and generation exchange (i.e. early weaning and artificial feeding) appear to be the successful strategies for the local eradication of bovine besnoitiosis. Adding to the already known mechanical transmission of B. besnoiti by blood-sucking flies, results of the present study suggest that secretophagous flies should also be evaluated as potential vectors of this coccidium species. more...

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., (Copyright © 2012 Australian Society for Parasitology Inc. All rights reserved.) more...

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. more...

The biology of Besnoitia besnoiti, the cause of bovine besnoitiosis, is poorly understood. Its definitive host is unknown, and information on potential intermediate hosts is scarce. In order to investigate potential definitive and intermediate hosts for European isolates of B. besnoiti, domestic dogs, cats, rabbits, guinea pigs (Cavia porcellus), gerbils (Meriones unguiculatus), common voles (Microtus arvalis) and NMRI-mice were inoculated with B. besnoiti isolated from naturally infected German cattle. Dogs and cats were fed 5×10(6)B. besnoiti tachyzoites (isolate Bb-GER1), or tissue cysts containing at least 2×10(7)B. besnoiti bradyzoites obtained from the skin of a naturally infected Limousin cow from the same herd where strain Bb-GER1 was isolated. Rodents and rabbits were subcutaneously inoculated with either 5×10(5) Bb-GER1 tachyzoites or 5×10(5) bradyzoites. Groups of 2-4 non-inoculated animals of each species were monitored as negative controls. Feces from all dogs and cats were daily examined by a sedimentation-flotation technique for at least 11 weeks after inoculation but no B. besnoiti oocysts were identified. Cats fed tachyzoites and dogs did not seroconvert, but specific antibodies to B. besnoiti tachyzoites were detected by IFAT (titer≥100) in 2 out of 3 cats fed tissue cysts since 5-7 weeks post infection. By immunoblot, these two cats exhibited a reaction pattern against tachyzoite antigens similar to that observed in naturally infected cattle. Antibodies against B. besnoiti tachyzoites were detected in all inoculated rodent species and rabbits by both, IFAT and immunoblot since 3 weeks post-inoculation. Rabbits and rodents, subcutaneously inoculated with same doses of inactivated bradyzoites remained serologically negative (IFAT titer<50). Clinical signs observed in the inoculated rabbits included fever, serous conjunctivitis and transient swelling of the testes. No clinical abnormalities were noticed in the other tested animal species. Voles developed pneumonia as observed by histological examination. B. besnoiti-DNA was detected by PCR in blood from rabbits, gerbils and voles at 9 days post-infection, and in skin, heart, lung, striated muscle and kidney tissues from voles at 19-21 weeks post-infection. Domestic dogs and cats could not be shown to be definitive hosts of B. besnoiti, but cats seroconverted after feeding on B. besnoiti tissue cysts indicating that B. besnoiti stages had invaded the cats' tissues. The molecular and serological results from this study indicate that European B. besnoiti isolates may infect cats, rabbits, guinea pigs, gerbils, mice and voles; however a persistence of the parasite could be demonstrated only in voles., (Copyright © 2011 Elsevier B.V. All rights reserved.) more...

Bovine besnoitiosis, an economically important disease in cattle in some countries of Africa and Asia, is emerging in Europe. The definitive host of Besnoitia besnoiti, the causative agent of bovine besnoitiosis, is unknown and the transmission of the parasite is not completely understood. Sensitive and quantitative DNA detection methods are needed to determine whether serologically positive animals are infectious and to examine the role of vectors (e.g. haematophagous insects) in the transmission of the parasite. To this end, we established two different 5'-nuclease quantitative assays to detect B. besnoiti infection in cattle and to estimate the parasite load in samples (BbRT1 and BbRT2). These PCRs are based on the sequence of the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene. Tests with serial dilutions of B. besnoiti genomic DNA in a buffer containing 100 ng/μl bovine DNA revealed a detection limit of 0.01 pg genomic B. besnoiti DNA. Reliable quantification was possible in samples containing ≥1 pg B. besnoiti genomic DNA with a coefficient of variation of ≤ 2%. To estimate the diagnostic sensitivity of the tests, skin biopsies and scrapings from the mucous membrane of the vestibulum vaginae (vaginal scrapings) were taken from cattle with clinical signs of chronic besnoitiosis. Regardless of the real time PCR assay used, 90.7% (39/43) of these animals were positive in at least one of two samples (skin or vaginal scrapings). Antibody titers, as determined by an immunofluorescent antibody test, and the threshold cycle values of the real time PCR obtained for skin samples and vaginal scrapings, were significantly correlated. The specificity of the PCRs was confirmed using genomic DNA from related parasites, including genomic DNA of Besnoitia spp., Neospora caninum, Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Isospora spp., Sarcocystis spp., Eimeria bovis, Cryptosporidium parvum, and Trypanosoma brucei brucei. Since the sequence of the ITS-1 region of B. besnoiti is identical with that of Besnoitia species isolated from donkeys (Besnoitia bennetti), and reindeer (Besnoitia tarandi), both real time PCRs detected also DNA of these parasites. One of the B. besnoiti real time PCRs, BbRT1, but not BbRT2, cross-reacted with Besnoitia darlingi, Besnoitia oryctofelisi, and Besnoitia neotomofelis when large amounts of genomic DNA (10 ng) were used. The other B. besnoiti real time PCR assay (BbRT2) was specific for B. besnoiti, B. bennetti and B. tarandi, but did not react when 10 ng DNA of other related parasite species from the genus Besnoitia or other genera were subjected to analysis., (Copyright © 2011 Elsevier B.V. All rights reserved.) more...

Bovine besnoitiosis is an economically important disease in cattle caused by the protozoan parasite Besnoitia besnoiti, which occurs endemically in many countries of Africa and Asia and is spreading in Europe. Serological identification of subclinically infected cattle is important to avoid the introduction of infected animals into naive herds. Here we determine the sensitivity and specificity of the PrioCHECK(®) Besnoitia Ab, a serological test recently introduced into the European market. Analytical specificity was examined using sera from animals experimentally infected with parasites related to B. besnoiti (n=27). Three animals experimentally infected with Neospora caninum or Toxoplasma gondii showed inconclusive reactions in the ELISA (percent positivity relative to the positive control [PP] 10% ≤ 20%) while all other sera reacted negative (PP<10%). An estimate of the diagnostic specificity was obtained by analysing field sera from bovine herds without besnoitiosis but with abortion problems associated to N. caninum (n=403). The analysis revealed a specificity of 94.3% or 96.8% depending on the applied cut-off (PP 10% or 20%, respectively). Sensitivity was assessed with sera from 110 animals of a herd in Germany where clinical bovine besnoitiosis was first diagnosed in September 2008. A positive serological reference standard was defined regarding sera from animals as reference positive, if these animals had tested positive in at least two of a panel of three other serological tests (two different B. besnoiti immunoblots and one immunofluorescence antibody test) on both of two sampling dates, November 2008 and April 2009. A diagnostic sensitivity of 91.8% or 75.5% was determined for sera collected in November 2008 and a sensitivity of 82.7% or 50% for sera collected in April 2009 (cut-off PP 10% or PP 20%, respectively). The marked drop in sensitivity from November 2008 to April 2009 was predominantly observed in reference-positive cattle without clinical signs. We conclude that PrioCHECK(®) Besnoitia Ab is a valuable diagnostic tool to detect clinically infected animals. Thus it may be used to support control measures, e.g., for the separation of infected animals from the remaining herd to avoid a further transmission of the infection within the herd., (Copyright © 2010 Elsevier B.V. All rights reserved.) more...

Besnoitia besnoiti, an apicomplexan parasite causes economically important disease in cattle in many countries of Africa and Asia is re-emerging in Europe. Serological identification of infected cattle is important because introduction of these animals into naive herds seems to play a major role in the transmission of the parasite. We report new, simplified immunoblot-based serological tests for the detection of B. besnoiti-specific antibodies. Antigens were used under non-reducing conditions in the immunoblots, because reduction of the antigen with beta-mercaptoethanol diminished the antigenicity in both, tachyzoites and bradyzoites. Ten B. besnoiti tachyzoite and ten bradyzoite antigens of 15-45 kDa molecular weight were recognized by B. besnoiti infected cattle, but not or only weakly detected by cattle infected with related protozoan parasites, Neospora caninum, Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, or Sarcocystis hirsuta. The sensitivity and specificity of B. besnoiti immunoblots were determined with sera from 62 German cattle with clinically confirmed besnoitiosis and 404 sera from unexposed German cattle including 214 sera from animals with a N. caninum-specific antibody response. Using a new scoring system, the highest specificity (100%) and sensitivity (90%) of the immunoblots were observed when reactivity to at least four of the ten selected tachyzoite or bradyzoite antigens was considered as positive. When a cut-off based on this scoring system was applied to both the tachyzoite- and the bradyzoite-based immunoblots, there was an almost perfect agreement with the indirect fluorescent antibody test with a titre of 200 as the positive cut-off. We identified and partially characterized 10 tachyzoite and 10 bradyzoite B. besnoiti antigens which may help to develop new specific and sensitive serological tests based on individual antigens and in the identification of possible vaccine candidates., (Copyright (c) 2010 Elsevier B.V. All rights reserved.) more...

Besnoitia besnoiti was in vitro isolated during the first recorded outbreak of bovine besnoitiosis in Germany. Molecular characterization of the new isolate, named Bb-GER1, revealed almost 100% identity with other B. besnoiti isolates obtained in Portugal, Spain, Israel or South Africa, when partial sequences of the 18S ribosomal RNA gene, of the internal transcribed spacer 1 and of the 5.8S RNA gene were compared. Cystozoites obtained from skin tissue of one bull were infectious for gamma-interferon knockout (GKO) mice by intraperitoneal (ip) inoculation. Tachyzoites were detected in the peritoneal cavity, spleen, liver and lung of the mice 5 days post-infection. The parasite could be maintained in GKO mice by ip inoculation for at least 5 passages. Peritoneal washings containing tachyzoites were obtained from infected mice and used to infect five cell lines (Vero, MARC-145, NA42/13, BHK(21), KH-R). The best growth of tachyzoites was observed in BHK(21) cells, but replication occurred to a smaller extent also in MARC-145, NA42/13 and KH-R cells. Subsequent comparative analyses revealed that after direct infection of these cell lines with cystozoites derived from bovine skin, the growth was best in NA42/13 cells. Considerable replication was also observed in the BHK(21) and KH-R cell lines. Our observations on the growth characteristics of Bb-GER1 partially contrast those for other isolates. The preferential growth in particular cell lines may be characteristic for particular B. besnoiti isolates. A potential association between growth properties and differences in virulence remains to be established. This is the first in vitro isolation of B. besnoiti from cattle in Germany. more...

Canine isolates of Hammondia heydorni from Argentina, Brazil, and the United States were analysed for genetic diversity. A total of 14 isolates were tested for their ability to produce amplification using three PCR assays, one targeting the common toxoplasmatiid ITS-1 region and 2 amplifying novel, H. heydorni-specific loci, HhAP7 and HhAP10. While the ITS-1 fragments could be amplified from all isolates, only six isolates were capable of amplifying the fragments from the novel loci. The PCR products were further investigated for genetic diversity using restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) techniques. Polymorphism in the digestion pattern was evident only at the HhAP10 locus, differentiating two of the Argentinean isolates from the remainder. Mobility shifts on SSCP gels revealed that the two Argentinean isolates were not only different from the other four isolates, but also differed from each other, both at the HhAP7 and HhAP10 loci. The ITS-1 fragments of all isolates were identical by RFLP. However, two distinct mobility patterns resulted when the products were electrophoresed on SSCP gels. Based on the sequence data from the ITS-1 and the two random loci, the isolates could be broadly classified into two distinct groups, within which minor polymorphisms were evident. In contrast, very little heterogeneity occurred in the sequences of corresponding ITS-1 regions of Neospora caninum and Toxoplasma gondii isolates. Thus, it is concluded that there is a considerable degree of microheterogeneity among isolates of H. heydorni. This diversity should be taken into consideration while attempting to elucidate the systematics, diagnostics, and biology of H. heydorni in relation to N. caninum. more...

Besnoitia sp. are apicomplexan coccidian parasites affecting several species of mammals and cold-blooded animals in several countries. Besnoitia sp. tissue cysts were seen in several tissues of five rabbits from a rabbit breeder in La Plata, Argentina. Bradyzoites released from macroscopic tissue cysts were inoculated onto bovine monocytes, and into interferon gamma gene knockout (KO) mice. Besnoitia sp. tachyzoites were seen in the peritoneal exudate of KO mice on day 10 pi and these tachyzoites were infective to other KO mice. Tachyzoites grown in cell culture were infective to gerbils (Meriones unguiculatus). This is the first report of Besnoitia sp. infection in any host in Argentina. more...