Effect of parasite dose and host age on the infection with Besnoitia besnoiti tachyzoites in cattle.

Bovine besnoitiosis is continuing to spread in Europe. Therefore, the development of ruminant animal models of infection is urgently needed to evaluate therapeutic and prophylactic tools. Herein, we studied the effect of parasite dose and host age on the infection dynamics with Besnoitia besnoiti tachyzoites in cattle in two independent experimental infections. In experiment A, twelve 3-month-old male calves were inoculated intravenously with either three different doses of tachyzoites (G1: 108 ; G2: 107 ; G3: 106 ) or with PBS (G4). In experiment B, six 14-month-old bulls were inoculated with 106 tachyzoites based on results obtained in experiment A. In both trials, clinical signs compatible with acute and chronic besnoitiosis were monitored daily; blood and skin samples were collected regularly for 70-115 days post-infection (pi). Finally, animals were killed, and tissues were collected for lesion and parasite detections. Infected animals developed mild-moderate signs compatible with acute besnoitiosis. Lymphadenopathy and fever were observed in both calves (from 12 hr until 7 days pi) and bulls (from 6 days until 9 days pi). Seroconversion was detected at 16-19 days pi, and antibody levels remained high. Infected animals did not developed characteristic clinical signs and macroscopic lesions of chronic besnoitiosis. However, successfully, parasite-DNA was detected in a reduced number of target tissues: conjunctiva, ocular sclera, epididymis, skin of the scrotum and carpus in calves (n = 10, 6 of which belonged to G3), and pampiniform plexus and testicular parenchyma in bulls. Remarkably, one tissue cyst and mild microscopic lesions were also detected. In summary, inoculated animals developed the acute besnoitiosis and chronic infection was evidenced by microscopic findings. However, our results suggest that tachyzoite dose and host age are not key variables for inducing clinical signs and macroscopic lesions characteristic of chronic besnoitiosis. Thus, a further refinement of this model should evaluate other parasite- and host-dependent variables.

Safety and efficacy of the bumped kinase inhibitor BKI-1553 in pregnant sheep experimentally infected with Neospora caninum tachyzoites.

Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlighted its relevance as an abortifacient in small ruminants. Vaccines or drugs for the control of neosporosis are lacking. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. We here present the pharmacokinetics, safety and efficacy of BKI-1553 in pregnant ewes and foetuses using a pregnant sheep model of N. caninum infection. BKI-1553 showed exposure in pregnant ewes with trough concentrations of approximately 4 µM, and of 1  µM in foetuses. Subcutaneous BKI-1553 administration increased rectal temperatures shortly after treatment, and resulted in dermal nodules triggering a slight monocytosis after repeated doses at short intervals. BKI-1553 treatment decreased fever in infected pregnant ewes already after two applications, resulted in a 37-50% reduction in foetal mortality, and modulated immune responses; IFNγ levels were increased early after infection and IgG levels were reduced subsequently. N. caninum was abundantly found in placental tissues; however, parasite detection in foetal brain tissue decreased from 94% in the infected/untreated group to 69-71% in the treated groups. In summary, BKI-1553 confers partial protection against abortion in a ruminant experimental model of N. caninum infection during pregnancy. In addition, reduced parasite detection, parasite load and lesions in foetal brains were observed.

Seroprevalence of Leptospirosis, Brucellosis, and Q Fever in a Wild Red Deer (Cervus elaphus) Population Kept in a Fenced Reserve in Absence of Contact with Livestock.

Wildlife health is of interest for public and animal health because wild animals have been identified as important sentinels for the surveillance for zoonotic pathogens. This work investigated Brucella spp., Coxiella burnetii, and Leptospira spp. infection seroprevalence in a free-ranging red deer population. The study was conducted in a fenced reserve with controlled hunting activity in central Spain with animals that did not have any contact with livestock. Sampling was performed at two time points before and 5 years after the implementation of new management measures, including a reduction in the red deer population in the reserve. In addition, the presence of Leptospira DNA was tested in placental and fetal samples from seropositive pregnant animals. Antibodies against Brucella and Coxiella were not detected in any sample. The seroprevalence of Leptospira was 9.4% (13/137) in the first sampling for serovars Canicola and Panama. Five years later, the prevalence rose to 38.5% (97/252) with Pomona, the only serovar detected. Animals older than 2 years (50%; 70/140) were more likely to be Pomona seropositive than animals ≤2 years old (25.2%; 27/107; p < 0.001). Leptospira DNA was not detected in any sample tested. In conclusion, wild red deer in this area without contact with livestock seem not to play an important role in Brucella spp. and C. burnetii maintenance. The high seroprevalence of Leptospira spp. serogroup Pomona could indicate a risk for people with narrow contact with these animals, but the carrier status was not assessed. Consequently, it is unknown if red deer would represent a risk for human infection. Considering that wild boar could be the source of infection to red deer, the role of wild boar in the spread of leptospirosis and the risk for human infection should be investigated.

The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress.

BACKGROUND: NTPases (also NTPDases) are enzymes with apyrase activity. They are widely distributed among eukaryotes, and also among members of the family Sarcocystidae. In Toxoplasma gondii, the TgNTPase accumulates in the dense granules, and has been commonly associated with the strain virulence. In the closely related Neospora caninum, the NcNTPase lacks nucleoside diphosphate hydrolase activity and appears to be more abundant in virulent isolates, indicating that it may contribute to the pathogenicity of neosporosis. However, so far no additional information on NcNTPase has been provided. METHODS: Herein, the NcNTPase coding sequences were analysed by different in silico and de novo sequencing approaches. A comparative analysis of NcNTPase and NcGRA7 in terms of protein dynamics, secretion, phosphorylation, and mRNA expression profiles during the tachyzoite lytic cycle was also carried out. Moreover, NcNTPase immunolocalization was analysed by confocal microscopy techniques over a set number of time-points. RESULTS: We describe the presence of three different loci containing three copies of the NcNTPase within the Nc-Liv genome, and report the existence of up to four different NcNTPase alleles in Nc-Liv. We also provide evidence for the occurrence of diverse protein species of the NcNTPase by two-dimensional gel electrophoresis. Both NcNTPase and NcGRA7 were similarly up-regulated and secreted during the egress and/or early invasion phases, and were phosphorylated. However, its secretion was not affected by the addition of calcium modulators such as A23187 and ethanol. NcNTPase and NcGRA7 localized in dense granules and parasitophorous vacuole membrane throughout the lytic cycle, although differed in their inmunolocalization during early invasion and egress. CONCLUSIONS: The present study reveals the complexity of the NcNTPase loci in N. caninum. We hypothesize that the expression of different isoforms of the NcNTPase protein could contribute to parasite virulence. Our findings showed regulation of expression, secretion and phosphorylation of NcNTPase suggesting a potential role for progression through the tachyzoites lytic cycle.

Besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates.

BACKGROUND: Bovine besnoitiosis, caused by the protozoan Besnoitia besnoiti, reduces productivity and fertility of affected herds. Besnoitiosis continues to expand in Europe and no effective control tools are currently available. Experimental models are urgently needed. Herein, we describe for the first time the kinetics of standardised in vitro models for the B. besnoiti lytic cycle. This will aid to study the pathogenesis of the disease, in the screening for vaccine targets and drugs potentially useful for the treatment of besnoitiosis. METHODS: We compared invasion and proliferation of one B. tarandi (from Finland) and seven B. besnoiti isolates (Bb-Spain1, Bb-Spain2, Bb-Israel, Bb-Evora03, Bb-Ger1, Bb-France, Bb-Italy2) in MARC-145 cell culture. Host cell invasion was studied at 4, 6, 8 and 24 h post infection (hpi), and proliferation characteristics were compared at 24, 48, 72, 96, 120, and 144 hpi. RESULTS: In Besnoitia spp., the key parameters that determine the sequential adhesion-invasion, proliferation and egress steps are clearly distinct from those in the related apicomplexans Toxoplasma gondii and Neospora caninum. Besnoitia spp. host cell invasion is a rather slow process, since only 50 % of parasites were found intracellular after 3-6 h of exposure to host cells, and invasion still took place after 24 h. Invasion efficacy was significantly higher for Bb-France, Bb-Evora03 and Bb-Israel. In addition, the time span for endodyogeny to take place was as long as 18-35 h. Bb-Israel and B. tarandi isolates were most prolific, as determined by the tachyzoite yield at 72 hpi. The total tachyzoite yield could not be predicted neither by invasion-related parameters (velocity and half time invasion) nor by proliferation parameters (lag phase and doubling time (dT)). The lytic cycle of Besnoitia was asynchronous as evidenced by the presence of three different plaque-forming tachyzoite categories (lysis plaques, large and small parasitophorous vacuoles). CONCLUSIONS: This study provides first insights into the lytic cycle of B. besnoiti isolates and a standardised in vitro model that allows screening of drug candidates for the treatment of besnoitiosis.

Neospora caninum tachyzoite immunome study reveals differences among three biologically different isolates.

Pathogenesis of bovine neosporosis is determined by different host- and parasite-dependent factors, including isolate virulence. A previous study identified that several Neospora caninum tachyzoite proteins were more abundant in virulent isolates, Nc-Liv and Nc-Spain7, compared with the low-virulent isolate Nc-Spain1H. Herein, we explored differences in the immunomes of these three isolates. Protein extracts from the Nc-Liv, Nc-Spain1H and Nc-Spain7 isolates were analysed in a 3×3 design by 2-DE immunoblot using sera from experimentally infected mice with these same three isolates. All isolates displayed a highly similar antigenic pattern when they were assessed using the same serum. Most of the reactive spots were located in the acidic region (pH 3-7) and grouped in 3 antigenic areas (250-70, 45-37 and 35-15 KDa). Differences found in the immunome depended on the sera used, regardless of the extract employed. In this sense, sera from Nc-Liv and Nc-Spain7 infected mice recognized the highest number of antigens, followed by Nc-Spain1H infected mice sera. In fact, 4 proteins identified by MS were not consistently detected in each isolate extract by sera from low-virulent Nc-Spain1H-infected mice: serine-threonine phosphatase 2C and superoxide dismutase (related to metabolism), gliding associated protein GAP45 (related to tachyzoites invasion), and NcGRA1 (located on dense granules). Similarly, 4 non-identified spots and another 2 spots chains located in 45-37 kDa area were not detected by this pooled sera. Variations between virulent Nc-Spain7 and Nc-Liv were limited to the absence of recognition by sera from Nc-Spain7-infected mice of GAP45 and the spot chains located in the 45-37 kDa area. These results suggest that variations in the immunome profiles rely on the immune response induced by each isolate and that these differentially recognized antigens could be investigated as putative virulence markers of neosporosis.

Prevalence of Besnoitia besnoiti infection in beef cattle from the Spanish Pyrenees.

Reports of recent outbreaks of bovine besnoitiosis in Europe have emphasized the need for prevalence studies to determine the importance of the disease. A previous cross-sectional study carried out in a northern province of Spain showed that seropositive cattle were beef breeds primarily located in regions where the disease is endemic (such as in the Pyrenees). The present study focused on this population. Sixty-three beef herds were selected and at least 50% of the animals in each herd were sampled (n = 3318). The herd, intra-herd and animal prevalence rates were calculated. All breeding bulls located in the same region were also sampled (n = 587), and the animal prevalence calculated. Sera were tested by ELISA. The herd prevalence rate of bovine besnoitiosis was 87.3%, and both sexes were similarly affected (approximately 50%). The results indicate that the disease is highly widespread in the Pyrenees, and that serological examination of cattle should be recommended when trading beef cattle.

First serosurvey of Besnoitia spp. infection in wild European ruminants in Spain.

Besnoitia besnoiti has been reported to affect cattle, wildebeest, kudu and impala, and B. tarandi other wild ruminants (caribou, reindeer, mule deer and musk ox), causing similar characteristic clinical signs and lesions. However, both Besnoitia species have been reported in different geographical areas and the link between the sylvatic and domestic life cycles of Besnoita spp. in wild ruminants and cattle remains unknown. The aim of this study was to evaluate the presence of specific antibodies against Besnoitia spp. in wild ruminants in Spain. A wide panel of sera from red deer (Cervus elaphus) (n=734), roe deer (Capreolus capreolus) (n=124), chamois (Rupicapra pyrenaica) (n=170) and mouflon (Ovis musimon) (n=20) collected from different locations of Spain was analyzed. Beef cattle were present in all sampled areas and, interestingly, bovine besnoitiosis has been widely reported in some of them (e.g., Pyrenees and Central Spain). Sera samples were first examined with an Enzyme-Linked Immunosorbent Assay (ELISA). For red deer and roe deer, the ELISA was standardized with positive and negative control sera from several Cervidae species (100% Se and 98% Sp). Chamois and mouflon sera samples were tested with a previously reported ELISA validated for bovine sera (97% Se and 95% Sp) using protein G as a conjugate. Positive results by ELISA were confirmed a posteriori with a tachyzoite-based Western blot. Sixty-one sera samples from red deer and 17 sera samples from roe-deer were seropositive or doubtful by ELISA. All samples from mouflon were seronegative and 15 sera samples from chamois were considered doubtful. B. besnoiti exposure was only confirmed clearly by Western blot in one red deer and one roe deer from the Spanish Pyrenees where the disease is traditionally endemic. This is the first serological report of Besnoitia spp. infection carried out in European wild ruminants and the results show that specific antibodies are present at least in red deer and roe-deer. Thus, wild ruminants from endemic regions of bovine besnoitiosis should be further studied because they may be putative reservoirs of the parasite.

Identification of Besnoitia besnoiti proteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis.

Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio ± 1.5, P<0.05 in t-test). Furthermore, 25 differentially expressed spots were selected and analysed by MALDI-TOF/MS. As a result, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.

First 2-DE approach towards characterising the proteome and immunome of Besnoitia besnoiti in the tachyzoite stage.

Bovine besnoitiosis is caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. It is considered to be a re-emergent disease in Europe and is also present in Africa and Asia. Due to the chronic and debilitating course of the disease, bovine besnoitiosis is responsible for severe economic losses. However, many aspects of the disease and parasite biology remain unknown. Proteomics studies could help to investigate relevant biological processes as well as host immune response associated with parasite infection. Both the proteome and immunome of the tachyzoite stage of B. besnoiti of the Bb-Spain1 isolate are described herein for the first time. Tachyzoite protein extracts were first separated by 2-DE SDS-PAGE using pH 3-10 NL IPG strips for Coomassie Brilliant Blue-stained gels and immunoblots. Eighty-five out of 265 spots visualised on Coomassie-stained gels were immunogenic when pooled serum from naturally infected cattle was used, and the distribution of immunogenic spots correlated with the 1-DE IDA pattern. Because most spots were found in the acidic range of the pH gradient, pH 3-6 L IPG strips were used next, and 58 out of 123 visualised spots proved to be immunogenic. Twenty-seven spots were identified by MALDI TOF/TOF to be 20 different proteins due to the presence of protein species. All proteins identified corresponded to highly conserved proteins among eukaryotes. Six proteins identified are related to energy metabolism, 3 are heat shock proteins, 4 proteins are related to host cell invasion processes, and 2 proteins are involved in cell redox homeostasis. A tryptophanyl tRNA synthetase, a putative gbp1p, nucleoredoxin, a putative receptor for activated C kinase, and a nuclear movement domain-containing protein were also identified. Among these proteins, fructose-1,6-bisphosphate aldolase, lactate dehydrogenase, pyruvate kinase, enolase, HSP60, HSP70, HSP90, actin and profilin proved to be immunogenic, and 5 were cross-reactive antigens between B. besnoiti and N. caninum. This first proteomic approach carried out in B. besnoiti should be followed by other studies to identify more specific parasite proteins.

Serological evidence of Besnoitia spp. infection in Canadian wild ruminants and strong cross-reaction between Besnoitia besnoiti and Besnoitia tarandi.

Bovine besnoitiosis, caused by Besnoitia besnoiti, is considered to be emergent in Europe and responsible for severe economic losses due to the chronic and debilitating course of the disease but has not been reported in North America. Besnoitia tarandi is a related species and it has been reported in reindeer and caribou from different locations of the Arctic Pole, including North America. Diagnosis of clinical besnoitiosis is largely based on the recognition of dermal grossly visible tissue cysts of Besnoitia. Nothing is known of cross reactivity between B. besnoiti and B. tarandi species. Here, we evaluated the use of serological tests employed in the diagnosis of bovine besnoitiosis for the detection of Besnoitia spp. infections in different wild ruminant species (caribou, elk, mule-deer, white-tailed deer, moose, muskox and bison) from Canada and investigated cross-reactivity between B. besnoiti and B. tarandi species by indirect immunofluorescence antibody test and Western blot. For this, species-specific antibodies were obtained in rabbits experimentally infected with B. besnoiti and B. tarandi. Marked cross reactivity was found between B. besnoiti and B. tarandi. For the first time, antibodies to Besnoitia spp. infection were found in 16 of 20 caribou (Ranginfer tarandus), seven of 18 muskox (Ovibos moschatus), one of three bison (Bison bison), but not in 20 elk (Cervus canadensis), 20 white tailed deer (Odocoileus virginianus), and 20 moose (Alces alces) in Canada; results were similar using B. besnoiti and B. tarandi as antigen. There was no cross reactivity between the two Besnoitia species, Neospora caninum and Toxoplasma gondii with the cut-offs applied that prevented to observe it. The present study provides evidence that the serological assays can be useful to accomplish large scale prevalence studies in caribou and other wildlife species. Further studies are needed to study sylvatic and domestic cycle of B tarandi and B. besnoiti.