Detección serológica y comparativa de Neosporosis, Leucosis y Brucelosis en bovinos del noreste de México

Tesis (Maestría en Ciencias Veterinarias con Especialidad en Salud Animal) UANL

Equine neosporosis: search for antibodies in cerebrospinal fluid and sera from animals with history of ataxia

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Seroprevalence and risk factors associated with neosporosis in sheep and dogs from farms

This study aimed to establish the seroprevalence and risk factors associated with neosporosis in sheep and dogs from rural properties. 1497 blood samples were collected from sheep and 42 from dogs that cohabited with sheep from 16 farms located in the central region of São Paulo State, Brazil. For the detection of N. caninum antibodies it was performed the indirect fluorescent antibody test (IFAT >= 25). For the epidemiological study it was applied a questionnaire for the owners or responsible from the sheep and dogs regarding informations related to neosporosis. The seroprevalence obtained out of the 1497 sheep sera tested was 8.0% (CI95% = 6.7-9.2%) and out of the 42 dogs 4.8% (CI95% = 0-7.2%). Variables statistically related to seropositivity for N. caninum in sheep were: dams well as water supply (P = 0.0004; OR = 2.15), presence of other domestic canids (P = 0.0013; OR = 2.38) and presence of reproductive problems (P = 0.0031; OR = 1.75). (C) 2011 Elsevier B.V. All rights reserved.

RecNcMIC3-1-R is a microneme- and rhoptry-based chimeric antigen that protects against acute neosporosis and limits cerebral parasite load in the mouse model for Neospora caninum infection

In order to achieve host cell entry, the apicomplexan parasite Neospora caninum relies on the contents of distinct organelles, named micronemes, rhoptries and dense granules, which are secreted at defined timepoints during and after host cell entry. It was shown previously that a vaccine composed of a mixture of three recombinant antigens, corresponding to the two microneme antigens NcMIC1 and NcMIC3 and the rhoptry protein NcROP2, prevented disease and limited cerebral infection and transplacental transmission in mice. In this study, we selected predicted immunogenic domains of each of these proteins and created four different chimeric antigens, with the respective domains incorporated into these chimers in different orders. Following vaccination, mice were challenged intraperitoneally with 2 × 10(6)N. caninum tachzyoites and were then carefully monitored for clinical symptoms during 4 weeks post-infection. Of the four chimeric antigens, only recNcMIC3-1-R provided complete protection against disease with 100% survivors, compared to 40-80% of survivors in the other groups. Serology did not show any clear differences in total IgG, IgG1 and IgG2a levels between the different treatment groups. Vaccination with all four chimeric variants generated an IL-4 biased cytokine expression, which then shifted to an IFN-γ-dominated response following experimental infection. Sera of recNcMIC3-1-R vaccinated mice reacted with each individual recombinant antigen, as well as with three distinct bands in Neospora extracts with similar Mr as NcMIC1, NcMIC3 and NcROP2, and exhibited distinct apical labeling in tachyzoites. These results suggest that recNcMIC3-1-R is an interesting chimeric vaccine candidate and should be followed up in subsequent studies in a fetal infection model.

Neospora caninum immunoblotting improves serodiagnosis of bovine neosporosis

Neospora caninum ranges among the major causes of infectious abortion in cattle worldwide. The present study was designed to improve the serodiagnostic tools by complementing a conventional ELISA with a highly sensitive and species-specific N. caninum immunoblot. To evaluate this test combination, sera from several groups of cows were tested. The first group, consisting of experimentally infected calves, showed that immunoblot antibody reactivities were detectable 1 to 3 days earlier than those found in ELISA. The first immunodominant bands that appeared were a 29-kDa (NcSAG1) and a 36-kDa (NcSRS2) antigen. Other groups, based upon naturally infected cattle, were used to compare the diagnostic sensitivity of ELISA and immunoblotting. Overall, N. caninum immunoblotting exhibited a higher sensitivity (98%) than ELISA (87%). Conversely, immunoblotting also confirm in two other cases, true transient negativation in some animals. In general, banding patterns and band staining intensity correlated to the semiquantitative ELISA findings. On the other hand, the banding pattern could not be used to discriminate between sera from animals with a recent abortion and those of cows with latent N. caninum infection. We also addressed putative cross-reactions due to infection with Toxoplasma gondii. Sera from animals with a serologically proven T. gondii infection were either clearly negative by Neospora immunoblotting or they yielded a specific immunoblot antibody profile indicating a double infection with N. caninum. Sera from animals with positive findings in both Toxoplasma and Neospora ELISA thus provided dichotomic results in the immunoblot by allowing to confirm or to rule out the specificity of the antibody reaction in Neospora ELISA. Altogether, our findings demonstrate that N. caninum immunoblotting is a very sensitive and specific complementary tool to improve the serology for N. caninum infections in cattle.

Influence of the gestational stage on the clinical course, lesional development and parasite distribution in experimental ovine neosporosis.

Neospora caninum is considered one of the main causes of abortion in cattle, yet recent studies have also emphasised its relevance as an abortifacient in small ruminants. In order to gain deeper insight into the pathogenesis of ovine neosporosis, pregnant ewes were intravenously inoculated with 10(6) tachyzoites of the Nc-Spain7 isolate at days 40, 90 or 120 of gestation. Infection during the first term resulted in the death of all foetuses between days 19 and 21 post-infection, showing mainly necrotic lesions in foetal liver and the highest parasite DNA detection and burden in both placenta and foetal viscera. After infection at day 90, foetal death was also detected in all ewes, although later (34-48 days post-infection). In this group, lesions were mainly inflammatory. Foetal livers showed the lowest frequency of lesions, as well as the lowest parasite detection and burden. All ewes infected at day 120 delivered viable lambs, although 3 out of 9 showed weakness and recumbency. Neospora DNA was detected in all lambs but one, and parasite burden was similar to that observed in day 90 group. Lesions in this group showed more conspicuous infiltration of inflammatory cells and higher frequency in foetal brain and muscle when compared to both previous groups. These results highlight the crucial role that the stage of gestation plays on the course of ovine neosporosis, similar to that reported in bovine neosporosis, and open the doors to consider sheep as a valid model for exogenous transplacental transmission for ruminant neosporosis.

A vaccine formulation combining rhoptry proteins NcROP40 and NcROP2 improves pup survival in a pregnant mouse model of neosporosis.

Currently there are no effective vaccines for the control of bovine neosporosis. During the last years several subunit vaccines based on immunodominant antigens and other proteins involved in adhesion, invasion and intracellular proliferation of Neospora caninum have been evaluated as targets for vaccine development in experimental mouse infection models. Among them, the rhoptry antigen NcROP2 and the immunodominant NcGRA7 protein have been assessed with varying results. Recent studies have shown that another rhoptry component, NcROP40, and NcNTPase, a putative dense granule antigen, exhibit higher expression levels in tachyzoites of virulent N. caninum isolates, suggesting that these could be potential vaccine candidates to limit the effects of infection. In the present work, the safety and efficacy of these recombinant antigens formulated in Quil-A adjuvant as monovalent vaccines or pair-wise combinations (rNcROP40+rNcROP2 and rNcGRA7+rNcNTPase) were evaluated in a pregnant mouse model of neosporosis. All the vaccine formulations elicited a specific immune response against their respective native proteins after immunization. Mice vaccinated with rNcROP40 and rNcROP2 alone or in combination produced the highest levels of IFN-γ and exhibited low parasite burdens and low IgG antibody levels after the challenge. In addition, most of the vaccine formulations were able to increase the median survival time in the offspring. However, pup survival only ensued in the groups vaccinated with rNcROP40+rNcROP2 (16.2%) and rNcROP2 (6.3%). Interestingly, vertical transmission was not observed in those survivor pups immunized with rNcROP40+rNcROP2, as shown by PCR analyses. These results show a partial protection against N. caninum infection after vaccination with rNcROP40+rNcROP2, suggesting a synergistic effect of the two recombinant rhoptry antigens.

Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.

Vaccines against neosporosis: what can we learn from the past studies?

Neospora caninum is an intracellular apicomplexan parasite, which is a leading cause of abortion in cattle; thus neosporosis represents an important veterinary health problem and is of high economic significance. The parasite can infect cattle via trans-placental transmission from an infected cow to its fetus (vertical transmission), or through the oral route via ingestion of food or water contaminated with oocysts that were previously shed with the feces of a canid definitive host (horizontal transmission). Although vaccination was considered a rational strategy to prevent bovine neosporosis, the only commercialized vaccine (Neoguard®) produced ambiguous results with relatively low efficacy, and was recently removed from the market. Therefore, there is a need to develop an efficient vaccine capable of preventing both, the horizontal transmission through infected food or water to a naïve animal as well as the vertical transmission from infected but clinically asymptomatic dams to the fetus. Different vaccine strategies have been investigated, including the use of live attenuated vaccines, killed parasite lysates, total antigens or antigen fractions from killed parasites, and subunit vaccines. The vast majority of experimental studies were performed in mice, and to a certain extent in gerbils, but there is also a large number of investigations that were conducted in cattle and sheep. However, it is difficult to directly compare these studies due to the high variability of the parameters employed. In this review, we will summarize the recent advances made in vaccine development against N. caninum in cattle and in mice and highlight the most important factors, which are likely to influence the degree of protection mediated by vaccination.

Repurposing of antiparasitic drugs: the hydroxy-naphthoquinone buparvaquone inhibits vertical transmission in the pregnant neosporosis mouse model.

The three anti-malarial drugs artemiside, artemisone, and mefloquine, and the naphthoquinone buparvaquone known to be active against theileriosis in cattle and Leishmania infections in rodents, were assessed for activity against Neospora caninum infection. All four compounds inhibited the proliferation of N. caninum tachyzoites in vitro with IC50 in the sub-micromolar range, but artemisone and buparvaquone were most effective (IC50 = 3 and 4.9 nM, respectively). However, in a neosporosis mouse model for cerebral infection comprising Balb/c mice experimentally infected with the virulent isolate Nc-Spain7, the three anti-malarial compounds failed to exhibit any activity, since treatment did not reduce the parasite burden in brains and lungs compared to untreated controls. Thus, these compounds were not further evaluated in pregnant mice. On the other hand, buparvaquone, shown earlier to be effective in reducing the parasite load in the lungs in an acute neosporosis disease model, was further assessed in the pregnant mouse model. Buparvaquone efficiently inhibited vertical transmission in Balb/c mice experimentally infected at day 7 of pregnancy, reduced clinical signs in the pups, but had no effect on cerebral infection in the dams. This demonstrates proof-of-concept that drug repurposing may lead to the discovery of an effective compound against neosporosis that can protect offspring from vertical transmission and disease.

Identificación y caracterización de las proteínas NcROP40 y NcNTPasa, y evaluación de su utilidad vacunal frente a la neosporosis

Neospora caninum es un parásito formador de quistes reconocido a nivel mundial como la principal causa de aborto en ganado vacuno, donde produce importantes pérdidas económicas. A pesar de que la vacunación se ha descrito como la estrategia de control más eficiente frente a la neosporosis bovina, hasta la fecha no existen formulaciones eficaces que prevengan la transmisión vertical y el aborto. En la actualidad, las medidas de control de la enfermedad dependen de un diagnóstico adecuado asociado a unas medidas de manejo concretas, por lo que el desarrollo de una vacuna frente a la neosporosis bovina es una tarea urgente. En este sentido, las vacunas de subunidades se presentan como una buena alternativa al uso de vacunas vivas, debido a su mayor seguridad y menor coste de producción; además, dichas vacunas pueden ser específicamente diseñadas frente a proteínas determinadas con el fin de bloquear procesos esenciales para la supervivencia del parásito. Desgraciadamente, el conocimiento de los mecanismos de invasión y proliferación de N. caninum es muy limitado a nivel molecular. Además, son pocos los estudios en los que se haya abarcado la identificación de factores de virulencia potenciales del parásito. Todo ello dificulta la selección de dianas apropiadas a la hora de diseñar nuevas formulaciones vacunales frente a la neosporosis...

Neospora caninum and reproductive wastage in extensively managed Queensland beef herds

Objective To compare reproduction in extensively managed, tropically adapted beef cows that were either seropositive or seronegative to Neospora caninum. Design Longitudinal study of cows within management groups. Methods Compare pregnancy with weaning outcomes for 502 seropositive and 3255 seronegative cows in 25 management groups. Results We found N. caninum in all herds, with an average of 20% of 2640 tested animals seropositive within management group; prevalence varied between 0% and 94%. At 7 of 10 sites assessed, there was evidence of horizontal transmission of N. caninum. There was no overall difference in pregnancy rate (79% vs 75%; P > 0.05), reproductive wastage after confirmed pregnancy diagnosis (11% vs 10%; P > 0.05) or weaning rate (67% vs 68%; P > 0.05) between seronegative and seropositive cows, respectively. In one herd where a combination of risk factors for N. caninum was present, a significant reduction in pregnancy rate occurred after the 6 months mating (85% vs 69%; P < 0.05). The fetal and calf losses observed were lowest in south-east Queensland (4.3% of 117 pregnancies), highest in north-west Queensland (15.5% of 413 pregnancies) and intermediate in north-east Queensland (10.2% of 1625 pregnancies). Other infectious agents that are known to cause reproductive wastage were endemic in many herds, though none appeared to cause significant fetal or calf loss in this study. Conclusion Despite a high prevalence of N. caninum, there was no apparent effect on beef cattle reproduction, but there is potential to cause reproductive wastage if known risk factors to neosporosis are in effect.

Pesquisa de anticorpos anti-Neospora caninum em bovinos leiteiros, cães e trabalhadores rurais da região Sudoeste do Estado de Mato Grosso

Considerando a importância da neosporose interferindo na produtividade animal, avaliou-se a frequência de anticorpos anti-Neospora caninum em amostras de soros de bovinos leiteiros da Região Sudoeste do Estado de Mato Grosso, Brasil, complementando-se com amostras sorológicas colhidas de cães e de humanos que conviviam nas mesmas propriedades rurais amostradas. Um total de 1.036 amostras de soros foram analisadas, sendo 932 de fêmeas bovinas leiteiras, 37 de cães e 67 de humanos, provenientes de 24 propriedades e examinados por meio da reação de imunofluorescência indireta (RIFI). As amostras de soros humanos reagentes foram testadas novamente por Western-blotting para confirmação dos resultados. Anticorpos anti-N. caninum foram encontrados em 499 bovinos (53,5%), em pelo menos um animal positivo por propriedade, em 25 caninos (67,6%) e em sete humanos (10,5%). Não houve diferença significativa no número de bovinos positivos por faixa etária. Os resultados obtidos indicam uma ampla disseminação de N. caninum na região estudada.