Essential oil supplementation in milk replacers: short- and long-term impacts on feed efficiency, the faecal microbiota and the plasma metabolome in dairy calves.

Early supplementation with oregano essential oil (EO) in milk replacer (MR) may improve growth, immune responses, the microbiota and the metabolome in dairy calves during pre-weaning and in adulthood. Sixteen female dairy calves (3 days of age) were divided in two groups (n = 8/group): the control group (no EO) and the EO group (0.23 ml of EO in MR during 45 days). After weaning, calves were kept in a feedlot and fed ad libitum. The animals were weighed, and blood and faecal samples were collected on days 3 (T0), 45 (T1) and 370 (T2) to measure the biochemical profile and characterise peripheral blood mononuclear cells (PBMCs; CD4+, CD8+, CD14+, CD21+ and WC1+), the metabolome and microbiota composition. The EO group only had greater average daily weight gain during the suckling (EO supplementation) period (P = 0.030). The EO group showed higher average CD14+ population (monocytes) values, a lower abundance of Ruminococcaceae UCG-014, Faecalibacterium, Blautia and Alloprevotella and increased abundances of Allistipes and Akkermansia. The modification of some metabolites in plasma, such as butyric acid, 3-indole-propionic acid and succinic acid, particularly at T1, are consistent with intestinal microbiota changes. The data suggest that early EO supplementation increases feed efficiency only during the suckling period with notable changes in the microbiota and plasma metabolome; however, not all of these changes can be considered desirable from a gut health point of view. Additional research studies is required to demonstrate that EOs are a viable natural alternative to antibiotics for improving calf growth performance and health.

Adjuvants influence the immune cell populations present at the injection site granuloma induced by whole-cell inactivated paratuberculosis vaccines in sheep.

Vaccination is the most effective tool for paratuberculosis control. Currently, available vaccines prevent the progression of clinical disease in most animals but do not fully protect them against infection and induce the formation of an injection site granuloma. The precise mechanisms that operate in response to vaccination and granuloma development, as well as the effect that adjuvants could trigger, have not been fully investigated. Therefore, this study aimed to investigate the injection site granulomas induced by two inactivated paratuberculosis vaccines, which differ in the adjuvant employed. Two groups of 45-day-old lambs were immunized with two commercially available vaccines-one (n = 4) with Gudair® and the other (n = 4) with Silirum®. A third group (n = 4) was not vaccinated and served as control. The peripheral humoral response was assessed throughout the study by a commercial anti-Mycobacterium avium subspecies paratuberculosis (Map) antibody indirect ELISA, and the cellular immune response was assessed similarly by the IFN-γ release and comparative intradermal tests. The injection site granulomas were measured during the experiment and sampled at 75 days post-vaccination (dpv) when the animals were euthanized. The tissue damage, antigen and adjuvant distribution, and the presence and amount of immune cells were then determined and assessed by immunohistochemical methods. Antibodies against Map antigens; a general macrophage marker (Iba1), M1 (iNOS), and M2 (CD204) macrophages; T (CD3), B (CD20), and γδ T lymphocytes, proteins MHC-II and NRAMP1, and cytokines IL-4, IL-10, TNF, and IFN-γ were employed. Silirum® elicited a stronger peripheral cellular immune response than Gudair®, while the latter induced larger granulomas and more tissue damage at the site of injection. Additionally, adjuvant and Map antigen distribution throughout the granulomatous inflammatory infiltrate, as well as the NRAMP1 cell expression, which is linked to antigen phagocytosis, were highly irregular. In Silirum® induced granulomas, a higher number of MHC-II and TNF-expressing cells and a lower number of M2 macrophages suggested an improved antigen presentation, which could be due to the better antigen distribution and reduced tissue damage induced by this vaccine.

Pathology of the Mammary Gland in Sheep and Goats.

The recognition of lesions of the mammary gland in small ruminants is a useful diagnostic procedure that can aid in the identification of several udder diseases. This article reviews the main pathological lesions in this organ in sheep and goats. Mastitis is, by far, the most commonly diagnosed change. Acute clinical mastitis is associated with bacterial infections, mainly Staphylococcus aureus or Mannheimia haemolytica. Lesions related to subclinical and chronic mastitis are also described, either as localized cases or as a part of systemic diseases such as contagious agalactia, maedi-visna or tuberculosis. Neoplasia is rare in the mammary gland of sheep and goats with sporadic mammary adenocarcinomas most commonly reported. Teat lesions, including those due to trauma, orf virus infection or papillomas, are predisposing factors for the subsequent development of mastitis.

Early response of monocyte-derived macrophages from vaccinated and non-vaccinated goats against in vitro infection with Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis is a disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Vaccination is the most cost-effective control method. However, despite the fact that macrophages are the main target cells for this pathogen, the precise mechanisms behind the response of the macrophage to Map infection and how it is modified by vaccination are yet poorly understood. The aim of this study was to investigate the effect of Silirum® vaccination in the early immune response of caprine monocyte-derived macrophages (CaMØs). Peripheral blood mononuclear cells (PBMCs) were obtained from vaccinated and non-vaccinated goats, cultured in vitro until differentiation to macrophages and infected with Map. After a 24 h incubation, Map viability and DNA were assessed in culture by viable colony count and real time quantitative polymerase chain reaction (qPCR). In addition, Map phagocytosis and expression of IL-10, IL-12, IFN-γ, TNF-α, IL-17A, IL-1ß, iNOS, IL-6 and MIP-1ß were also evaluated through immunofluorescence labelling and reverse transcriptase qPCR (RT-qPCR), respectively. A significant reduction of Map viability was observed in both supernatants (P < 0.05) and CaMØs (P < 0.001) from the vaccinated group. Similarly, the percentage of infected CaMØs and the number of internalized Map by CaMØs (P < 0.0001) was higher in the vaccinated group. Finally, iNOS (P < 0.01) and IL-10 were significantly up-regulated in CaMØs from vaccinated goats, whereas only MIP-1ß was up-regulated in non-vaccinated animals (P < 0.05). These results show that vaccination modifies the immune response of CaMØs, suggesting that the phagocytosis and microbiocidal activity of macrophages against Map is enhanced after vaccination.

Influence of Heterologous and Homologous Vaccines, and Their Components, on the Host Immune Response and Protection Against Experimental Caprine Paratuberculosis.

Vaccination against paratuberculosis, a chronic disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map), has been considered as the most effective control method. However, protection is incomplete, and the mechanisms operating in the response of the animals to vaccination are not fully understood. Therefore, this study analyzed the immune response and the effects on protection against Map infection, elicited by paratuberculosis (Silirum®) and tuberculosis (heat-inactivated M. bovis [HIMB]) vaccines and their components in a caprine experimental model. Fifty goat kids were divided into 10 groups (n = 5) according to their vaccination (Silirum®, HIMB and nonvaccinated), immunization (inactivated bacteria or adjuvant), and/or infection. Oral challenge with Map was performed 45 days postvaccination/immunization (dpv), and animals were euthanized at 190 dpv. Peripheral immune response and proportion of lymphocyte subpopulations were assessed monthly by enzyme-linked immunosorbent assay and flow cytometry analysis, respectively. Local immune response, proportion of tissue lymphocyte subpopulations, Map detection (polymerase chain reaction), and histological examination were conducted in gut-associated lymphoid tissues. All infected groups developed paratuberculosis granulomatous lesions despite vaccination or immunization. The Silirum® and HIMB-vaccinated groups showed a considerable lesion reduction consistent with a significant peripheral cellular and humoral immune response. Besides, a lower number of granulomas were observed in groups immunized with inactivated bacteria and adjuvants in comparison to nonvaccinated and infected group. However, despite not being significant, this reduction was even higher in adjuvant immunized groups, which developed milder granulomatous lesion with no detectable peripheral immune responses associated with immunization. No changes in the peripheral and local proportion of lymphocyte subsets or local immune response were detected in relation to either vaccination/immunization or infection. Despite that paratuberculosis and tuberculosis vaccination showed a partial and cross-protection against Map infection, respectively, only histological examination could assess the progression of infection in these animals. In addition, the pattern observed in the reduction of the lesions in adjuvant immunized groups suggests the possible involvement of a nonspecific immune response that reduces the development of granulomatous lesions.

Optimized in vitro isolation of different subpopulation of immune cells from peripheral blood and comparative techniques for generation of monocyte-derived macrophages in small ruminants.

Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.

Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks.

BACKGROUND: Toxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spain. METHODS: Selected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes. RESULTS: As a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pattern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms. CONCLUSIONS: The present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversity.

Crosstalk between Neospora caninum and the bovine host at the maternal-foetal interface determines the outcome of infection.

Neospora caninum is an apicomplexan cyst-forming parasite that is considered one of the main causes of abortion. The pathogenic mechanisms associated with parasite virulence at the maternal-foetal interface that are responsible for the outcome of infection are largely unknown. Here, utilizing placentomes from cattle experimentally infected with high-virulence (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates, we studied key elements of the innate and adaptive immune responses, as well as components of the extracellular matrix (ECM), at 10 and 20 days post-infection (dpi). The low-virulence isolate elicited a robust immune response characterized by upregulation of genes involved in pathogen recognition, chemokines and pro-inflammatory cytokines, crucial for its adequate control. In addition, Nc-Spain1H triggered the expression of anti-inflammatory cytokines and other mechanisms implicated in the maintenance of ECM integrity to ensure foetal survival. In contrast, local immune responses were initially (10 dpi) impaired by Nc-Spain7, allowing parasite multiplication. Subsequently (20 dpi), a predominantly pro-inflammatory Th1-based response and an increase in leucocyte infiltration were observed. Moreover, Nc-Spain7-infected placentomes from animals carrying non-viable foetuses exhibited higher expression of the IL-8, TNF-α, iNOS and SERP-1 genes and lower expression of the metalloproteases and their inhibitors than Nc-Spain7-infected placentomes from animals carrying viable foetuses. In addition, profound placental damage characterized by an alteration in the ECM organization in necrotic foci, which could contribute to foetal death, was found. Two different host-parasite interaction patterns were observed at the bovine placenta as representative examples of different evolutionary strategies used by this parasite for transmission to offspring.

Vascular wall injury and inflammation are key pathogenic mechanisms responsible for early testicular degeneration during acute besnoitiosis in bulls.

BACKGROUND: Bovine besnoitiosis, caused by the apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that notably impairs fertility. Acutely infected bulls may develop respiratory signs and orchitis, and sterility has been reported in chronic infections. However, the pathogenesis of acute disease and its impact on reproductive function remain unknown. METHODS: Herein, we studied the microscopic lesions as well as parasite presence and load in the testis (pampiniform plexus, testicular parenchyma and scrotal skin) of seven bulls with an acute B. besnoiti infection. Acute infection was confirmed by serological techniques (IgM seropositive results and IgG seronegative results) and subsequent parasite detection by PCR and histological techniques. RESULTS: The most parasitized tissue was the scrotal skin. Moreover, the presence of tachyzoites, as shown by immunohistochemistry, was associated with vasculitis, and three bulls had already developed juvenile tissue cysts. In all animals, severe endothelial injury was evidenced by marked congestion, thrombosis, necrotizing vasculitis and angiogenesis, among others, in the pampiniform plexus, testicular parenchyma and scrotal skin. Vascular lesions coexisted with lesions characteristic of a chronic infection in the majority of bulls: hyperkeratosis, acanthosis and a marked diffuse fibroplasia in the dermis of the scrotum. An intense inflammatory infiltrate was also observed in the testicular parenchyma accompanied by different degrees of germline atrophy in the seminiferous tubules with the disappearance of various strata of germ cells in four bulls. CONCLUSIONS: This study confirmed that severe acute besnoitiosis leads to early sterility that might be permanent, which is supported by the severe lesions observed. Consequently, we hypothesized that testicular degeneration might be a consequence of (i) thermoregulation failure induced by vascular lesions in pampiniform plexus and scrotal skin lesions; (ii) severe vascular wall injury induced by the inflammatory response in the testis; and (iii) blood-testis barrier damage and alteration of spermatogenesis by immunoresponse.

Endogenous transplacental transmission of Neospora caninum during successive pregnancies across three generations of naturally infected sheep.

Endogenous transplacental transmission, which occurs during pregnancy as the result of reactivation of a latent infection in the dam, is the main mechanism of propagation of Neospora caninum within cattle herds. However, the importance of this propagation mechanism has not yet been evaluated in relation to ovine neosporosis. In this study, involving three generations of ewes naturally infected by N. caninum, we demonstrated that endogenous transplacental transmission may also be highly efficient in the ovine host since transmission of infection occurred in 96.6% of gestations and the congenital infection rate ranged between 66.7 and 93%. Nevertheless, parasite burdens decreased gradually in consecutive generations. Reactivation of latent infections had a strong impact on the pregnancy outcome, with high mortality rates recorded in the offspring of the two first generations of ewes (21.4-46.1%). Histological examination of the brain revealed that all aborted foetuses had characteristic lesions of neosporosis (necrotic glial foci) and a few parasite cysts, whereas most stillborn and newborn lambs that died shortly after birth had non-specific lesions (mild glial foci without necrosis) and parasite cysts were more frequent. Microsatellite analysis revealed scarce genetic variability in the N. caninum population, in accordance with a scenario in which infections were of a single origin and were exclusively maintained by clonal propagation through endogenous transplacental transmission.

Exposure to Neospora spp. and Besnoitia spp. in wildlife from Israel.

Neosporosis and besnoitiosis, caused by cyst-forming protozoa Neospora caninum and Besnoitia besnoiti, respectively, are parasitic infestations of livestock in Israel. These parasites cause significant economic losses in cattle due to reproductive and productive disorders. Both parasites have been detected in several wild ruminant species throughout other regions of the world, while the existence of a sylvatic life cycle in Israel remains uncertain. Thus, a wide panel of 871 sera from two wild carnivores and nine wild ruminant species were tested. All sera were first analysed by MAT for an initial screening and positive samples were confirmed a posteriori by Western blot. Additionally, a complementary IFAT was used for the detection of antibodies against N. caninum. Neospora antibodies were present in six out of the 11 species investigated, whereas Besnoitia antibodies were undetected. Golden jackal, red fox, addax, Arabian oryx, Persian fallow deer, mouflon, mountain gazelle, Nubian ibex, scimitar horned oryx and water buffalo were seropositive against N. caninum infection by IFAT and/or MAT. Moreover, the presence of Neospora spp.-specific antibodies was confirmed by Western blot in golden jackal (6/189; 3.2%), red fox (1/75; 1.3%), Persian fallow deer (13/232; 5.6%), mouflon (1/15; 16.7%), Nubian ibex (22/55; 40%) and water buffalo (12/18; 66.7%). Addax (1/49) and water buffalo (1/18) were MAT-seropositive against B. besnoiti but were seronegative by Western blot. Hence, Neospora sylvatic cycle is present in Israel and may cross over to a domestic life cycle. In contrast, wildlife species investigated are unlikely to present a risk of transmitting Besnoitia to livestock in Israel.

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.

Virulence in Mice of a Toxoplasma gondii Type II Isolate Does Not Correlate With the Outcome of Experimental Infection in Pregnant Sheep.

Toxoplasma gondii is an apicomplexan parasite that infects almost all warm-blooded animals. Little is known about how the parasite virulence in mice extrapolates to other relevant hosts. In the current study, in vitro phenotype and in vivo behavior in mice and sheep of a type II T. gondii isolate (TgShSp1) were compared with the reference type II T. gondii isolate (TgME49). The results of in vitro assays and the intraperitoneal inoculation of tachyzoites in mice indicated an enhanced virulence for the laboratory isolate, TgME49, compared to the recently obtained TgShSp1 isolate. TgShSp1 proliferated at a slower rate and had delayed lysis plaque formation compared to TgME49, but it formed more cyst-like structures in vitro. No mortality was observed in adult mice after infection with 1-105 tachyzoites intraperitoneally or with 25-2,000 oocysts orally of TgShSp1. In sheep orally challenged with oocysts, TgME49 infection resulted in sporadically higher rectal temperatures and higher parasite load in cotyledons from ewes that gave birth and brain tissues of the respective lambs, but no differences between these two isolates were found on fetal/lamb mortality or lesions and number of T. gondii-positive lambs. The congenital infection after challenge at mid-pregnancy with TgShSp1, measured as offspring mortality and vertical transmission, was different depending on the challenged host. In mice, mortality in 50% of the pups was observed when a dam was challenged with a high oocyst dose (500 TgShSp1 oocysts), whereas in sheep infected with the same dose of oocysts, mortality occurred in all fetuses. Likewise, mortality of 9 and 27% of the pups was observed in mice after infection with 100 and 25 TgShSp1 oocysts, respectively, while in sheep, infection with 50 and 10 TgShSp1 oocysts triggered mortality in 68 and 66% of the fetuses/lambs. Differences in vertical transmission in the surviving offspring were only found with the lower oocyst doses (100% after infection with 10 TgShSp1 oocysts in sheep and only 37% in mice after infection with 25 TgShSp1 oocysts). In conclusion, virulence in mice of T. gondii type II isolates may not be a good indicator to predict the outcome of infection in pregnant sheep.

Advances in the diagnosis of bovine besnoitiosis: current options and applications for control.

Bovine besnoitiosis, which is caused by the tissue cyst-forming intracellular parasite Besnoitia besnoiti, is a chronic and debilitating disease that is responsible for severe economic losses in the cattle raised under extensive husbandry systems. The absence of vaccines, treatments or a health scheme at local, national and international levels has led to a rapid spread of bovine besnoitiosis from western Europe towards eastern countries and northwards. Moreover, this parasitic disease is widely present in many sub-Saharan countries. Thus, bovine besnoitiosis should be included in the animal health scheme of beef cattle herds. Accurate diagnostic tools and common diagnostic procedures are mandatory in any control programme. Relevant advances have been made in this field during the last decade. Succeeding with accurate diagnosis relies on the technique employed and the antibody and parasite kinetics of the infection stage, which may notably influence control programmes and surveillance. Moreover, control programmes should be adapted to the epidemiological status of the disease, as the disease presentation in a herd has important implications for prospective control. Herein, we review the clinical disease presentation of bovine besnoitiosis and the correlation between its clinical course and laboratory parameters. We also provide an update on the available diagnostic tools, discuss their strengths and pitfalls, and provide guidelines for their use in control, surveillance and epidemiological studies. A rational control strategy is also recommended.

A serosurvey of selected cystogenic coccidia in Spanish equids: first detection of anti-Besnoitia spp. specific antibodies in Europe.

BACKGROUND: Equine besnoitiosis, caused by Besnoitia bennetti, and equine protozoal myeloencephalitis (EPM), caused by Sarcocystis neurona and Neospora hughesi are relevant equine diseases in the Americas that have been scarcely studied in Europe. Thus, a serosurvey of these cystogenic coccidia was carried out in Southern Spain. A cross-sectional study was performed and serum samples from horses (n = 553), donkeys (n = 85) and mules (n = 83) were included. An in-house enzyme-linked immunosorbent assay (ELISA) was employed to identify a Besnoitia spp. infection and positive results were confirmed by an a posteriori western blot. For Neospora spp. and Sarcocystis spp., infections were detected using in-house ELISAs based on the parasite surface antigens N. hughesi rNhSAG1 and S. neurona rSnSAG2/3/4. Risk factors associated with these protozoan infections were also investigated. RESULTS: Antibodies against Besnoitia spp., Neospora spp. and Sarcocystis spp. infections were detected in 51 (7.1%), 46 (6.4%) and 20 (2.8%) of 721 equids, respectively. The principal risk factors associated with a higher seroprevalence of Besnoitia spp. were the host species (mule or donkey), the absence of shelter and the absence of a rodent control programme. The presence of rodents was the only risk factor for Neospora spp. infection. CONCLUSIONS: This study was the first extensive serosurvey of Besnoitia spp. infection in European equids accomplished by two complementary tests and gives evidence of the presence of specific antibodies in these populations. However, the origin of the infection is still unclear. Further parasite detection and molecular genotyping are needed to identify the causative Besnoitia and Neospora species. Finally, cross-reactions with antibodies directed against other species of Sarcocystis might explain the positive reactions against the S. neurona antigens.

Characterization of an outbreak of emerging bovine besnoitiosis in southwestern Spain.

Bovine besnoitiosis is an emerging disease in Europe, presenting quick spread toward central and southern Spain. Characterization of an outbreak in a free-ranging Limousin and Avileña beef cattle herd from southwestern Spain territories is attempted. Serological survey in the herd revealed increase of number of infected animals, from 34.3 % on first diagnoses/exams on December 2013 to 42.5 % in the second on April 2014. Blood analysis and serum biochemistry showed important alterations like leukocytosis (+33.2 % of mean value), with lymphocytosis (+205.3 %) and increase of LDH (+25.1 %), associated with tissue damage. Clinical cases were only observed in Limousin animals. Along with typical lesions of acute and chronic besnoitiosis, inflammatory and degenerative processes and parasitic cysts were present in the corpus cavernosum and the corpus spongiosum of penis. By using polymerase chain reaction (PCR) sequencing of 18S rDNA, Besnoitia besnoiti was confirmed as causative agent; microsatellite sequence analyses showed the homology of isolates with previously studied strains.

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.

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.