Surgical and postsurgical management of abdominal paragangliomas and pheochromocytomas.

PURPOSE: To describe our current protocol for surgical and postsurgical management of abdominal paragangliomas (PGLs) and pheochromocytomas, with a special focus on multidisciplinary management in centres with experience. METHODS: The physicians involved in the management of patients with abdominal PGLs and pheochromocytomas of our hospital reviewed systematically current knowledge on the surgical management of abdominal PGLs and pheochromocytomas. RESULTS: Currently, surgery is considered the treatment of choice for abdominal PGLs and pheochromocytomas. The choice of surgical approach is determined based on the location of the lesion, size, patient́s body habitus and the likelihood of malignancy. Laparoscopic surgery is usually considered the gold standard approach for pheochromocytomas, but open access should be considered in invasive and/or potentially malignant tumours >8-10 cm and for abdominal PGLs. Postsurgical management of pheochromocytomas and PGLs includes close hemodynamic monitoring and treatment of postsurgical complications, the pathological study of the surgical specimen, reassessment of hormonal and/or radiological status and planning of follow-up based on the risk of recurrence and malignancy. CONCLUSION: Surgery represents the treatment of choice of most abdominal PGLs and pheochromocytomas. Optimal postsurgical evaluation, including hemodynamic, pathological, hormonal, and radiological evaluation, should be performed by a multidisciplinary team specializing in PGL/pheochromocytoma management.

Manejo quirúrgico y posquirúrgico de paragangliomas abdominales y feocromocitomas / Surgical and postsurgical management of abdominal paragangliomas and pheochromocytomas

Objetivo Describir nuestro protocolo actual para el manejo quirúrgico y posquirúrgico de los paragangliomas abdominales (PGL) y los feocromocitomas, con especial atención en el manejo multidisciplinar en centros con experiencia. Métodos Los facultativos implicados en el tratamiento de pacientes con PGL abdominales y feocromocitomas de nuestro hospital revisaron sistemáticamente la evidencia científica actualmente disponible sobre el tratamiento quirúrgico de los PGL abdominales y feocromocitomas. Resultados La cirugía se considera el tratamiento de primera elección para los PGL abdominales y feocromocitomas. La decisión sobre el tipo de abordaje quirúrgico se basa en la localización y el tamaño de la lesión, la constitución corporal del paciente y la probabilidad estimada de malignidad. La cirugía laparoscópica suele considerarse el abordaje de referencia para los feocromocitomas, pero en los tumores invasivos y/o potencialmente malignos de más de 8-10 cm y en los PGL abdominales debe considerarse el abordaje abierto. El tratamiento posquirúrgico de los feocromocitomas y los PGL incluye una monitorización hemodinámica estrecha, el tratamiento de las complicaciones posoperatorias, el estudio patológico de la muestra quirúrgica, la reevaluación del estado hormonal y/o radiológico y la planificación del seguimiento en función del riesgo de recurrencia y malignidad. Conclusión La cirugía representa el tratamiento de elección de la mayoría de los PGL abdominales y feocromocitomas. La evaluación posoperatoria óptima, que incluye la evaluación hemodinámica, patológica, hormonal y radiológica, debe ser realizada por un equipo multidisciplinar especializado en el tratamiento de PGL/feocromocitomas (AU)

Purpose To describe our current protocol for surgical and postsurgical management of abdominal paragangliomas (PGLs) and pheochromocytomas, with a special focus on multidisciplinary management in centres with experience. Methods The physicians involved in the management of patients with abdominal PGLs and pheochromocytomas of our hospital reviewed systematically current knowledge on the surgical management of abdominal PGLs and pheochromocytomas. Results Currently, surgery is considered the treatment of choice for abdominal PGLs and pheochromocytomas. The choice of surgical approach is determined based on the location of the lesion, size, patient́s body habitus and the likelihood of malignancy. Laparoscopic surgery is usually considered the gold standard approach for pheochromocytomas, but open access should be considered in invasive and/or potentially malignant tumours > 8-10 cm and for abdominal PGLs. Postsurgical management of pheochromocytomas and PGLs includes close hemodynamic monitoring and treatment of postsurgical complications, the pathological study of the surgical specimen, reassessment of hormonal and/or radiological status and planning of follow-up based on the risk of recurrence and malignancy. Conclusion Surgery represents the treatment of choice of most abdominal PGLs and pheochromocytomas. Optimal postsurgical evaluation, including hemodynamic, pathological, hormonal, and radiological evaluation, should be performed by a multidisciplinary team specializing in PGL/pheochromocytoma management (AU)

A frequency comb stabilized Ti:Sa laser as a self-reference for ion-trap experiments with a 40Ca+ ion.

In this study, we report on the stabilization of a continuous-wave Ti:Sa laser to an optical frequency comb. The laser is emitting at 866 nm to address one of the transitions required for Doppler cooling of a single 40Ca+ ion in a linear Paul trap (2D3/2 ↔P1/22). The stabilized Ti:Sa laser is utilized to calibrate an ultra-accurate wavelength meter. We certify this self-reference laser source by comparing the results from monitoring the laser-cooled 40Ca+ ion in the linear Paul trap, with those obtained when a HeNe laser is used for calibration. The use of this self-reference is compatible with the simultaneous use of the comb for precision spectroscopy in the same ion-trap experiment.

Results from the INMUNOSUN-SOGUG trial: a prospective phase II study of sunitinib as a second-line therapy in patients with metastatic renal cell carcinoma after immune checkpoint-based combination therapy.

BACKGROUND: The INMUNOSUN trial had the objective of prospectively evaluating the efficacy and safety of sunitinib as a pure second-line treatment in patients with metastatic renal cell carcinoma (mRCC) who have progressed to first-line immune checkpoint inhibitor (ICI)-based therapies. PATIENTS AND METHODS: A multicenter, phase II, single-arm, open-label study was carried out in patients with a histologically confirmed diagnosis of mRCC with a clear-cell component who had progressed to a first-line regimen of ICI-based therapies. All patients received sunitinib 50 mg once daily orally for 4 weeks, followed by a 2-week rest period following package insert instructions. The primary outcome was the objective response rate. RESULTS: Twenty-one assessable patients were included in the efficacy and safety analyses. Four patients [19.0%, 95% confidence interval (CI) 2.3% to 35.8%] showed an objective response (OR), and all of them had partial responses. Additionally, 14 (67%) patients showed a stable response, leading to clinical benefit in 18 patients (85.7%, 95% CI 70.7% to 100%). Among the four assessable patients who showed an OR, the median duration of the response was 7.1 months (interquartile range 4.2-12.0 months). The median progression-free survival (PFS) was 5.6 months (95% CI 3.1-8.0 months). The median overall survival (OS) was 23.5 months (95% CI 6.3-40.7 months). Patients who had better antitumor response to first-line ICI-based treatment showed a longer PFS and OS with sunitinib. The most frequent treatment-emergent adverse events were diarrhea (n = 11, 52%), dysgeusia (n = 8, 38%), palmar-plantar erythrodysesthesia (n = 8, 38%), and hypertension (n = 8, 38%). There was 1 patient who exhibited grade 5 pancytopenia, and 11 patients experienced grade 3 adverse events. Eight (38%) patients had serious adverse events, four of which were considered to be related to sunitinib. CONCLUSION: Although the INMUNOSUN trial did not reach the pre-specified endpoint, it demonstrated that sunitinib is active and can be safely used as a second-line option in patients with mRCC who progress to new standard ICI-based regimens.

Coherent manipulation of an Andreev spin qubit.

Two promising architectures for solid-state quantum information processing are based on electron spins electrostatically confined in semiconductor quantum dots and the collective electrodynamic modes of superconducting circuits. Superconducting electrodynamic qubits involve macroscopic numbers of electrons and offer the advantage of larger coupling, whereas semiconductor spin qubits involve individual electrons trapped in microscopic volumes but are more difficult to link. We combined beneficial aspects of both platforms in the Andreev spin qubit: the spin degree of freedom of an electronic quasiparticle trapped in the supercurrent-carrying Andreev levels of a Josephson semiconductor nanowire. We performed coherent spin manipulation by combining single-shot circuit-quantum-electrodynamics readout and spin-flipping Raman transitions and found a spin-flip time T S = 17 microseconds and a spin coherence time T 2E = 52 nanoseconds. These results herald a regime of supercurrent-mediated coherent spin-photon coupling at the single-quantum level.

Protocol for presurgical and anesthetic management of pheochromocytomas and sympathetic paragangliomas: a multidisciplinary approach.

OBJECTIVE: To offer a practical guide for the presurgical and anesthetic management of pheochromocytomas and sympathetic paragangliomas (PGLs). METHODS: This protocol was based on a comprehensive review of the literature and on our own multidisciplinary team's experience from managing pheochromocytoma and sympathetic PGLs at a referral center. RESULTS: Patients with pheochromocytomas and sympathetic paragangliomas (PGLs) may develop potentially life-threatening complications, especially during surgical procedures. A complete biochemical, radiological, genetic, and cardiological assessment is recommended in the preoperative stage as it provides an evaluation of the risk of surgical complications and malignancy, allowing individualization of the presurgical treatment. Treatment with α-blockade and proper volume expansion in the preoperative stage significantly reduces the perioperative morbidity. During surgery, the anesthesiologist should look for a deep anesthetic level that inhibits the cardiovascular effects of catecholamines to minimize the risk of intraoperative complications. CONCLUSIONS: An optimal presurgical evaluation of pheochromocytomas/ sympathetic PGL requires a multidisciplinary approach, including a complete hormonal, radiological, cardiac, genetic, and functioning evaluation in most cases. A proper preoperative evaluation in combination with strict blood pressure and heart rate control, and blood volume status optimization, will significantly reduce the risk of intraoperative and perioperative complications. In those patients who unfortunately develop intraoperative complications, the role of the anesthesiologist is essential since the selection of the appropriate management has a direct impact on morbimortality reduction.

Low Field Nano-NMR via Three-Level System Control.

Conventional control strategies for nitrogen-vacancy centers in quantum sensing are based on a two-level model of their triplet ground state. However, this approach fails in regimes of weak bias magnetic fields or strong microwave pulses, as we demonstrate. To overcome this limitation, we propose a novel control sequence that exploits all three levels by addressing a hidden Raman configuration with microwave pulses tuned to the zero-field transition. We report excellent performance in typical dynamical decoupling sequences, opening up the possibility for nano-NMR operation in low field environments.

Development and characterization of monoclonal antibodies against Besnoitia besnoiti tachyzoites.

This is the first report on the development and characterization of eight monoclonal antibodies (MABs) generated against whole- and membrane-enriched tachyzoite extracts of the apicomplexan parasite Besnoitia besnoiti. Confocal laser scanning immunofluorescence microscopy was used to localize respective epitopes in B. besnoiti tachyzoites along the lytic cycle. A pattern compatible with dense granule staining was observed with MABs 2.A.12, 2.F.3 and 2.G.4, which could be confirmed by immunogold electron microscopy for MABs 2.A.12 and 2.F.3. In particular, MABs 2.F.3 and 2.G.4 were secreted during early invasion, proliferation and egress phases. MABs 3.10.8 and 5.5.11 labelled the tachyzoite surface, whilst MABs 1.17.8, 8.9.2 and 2.G.A recognized the apical tip, which is reminiscent for microneme localization. Besides, the epitopes recognized by the latter two (MABs 8.9.2 and 2.G.A) exhibited a redistribution from the anterior part across the parasite surface towards the posterior end during invasion. Most MABs developed were genus-specific. Indeed, the MABs cross-reacted neither with T. gondii nor with N. caninum tachyzoites. In summary, we have generated MABs that will be useful to study the key processes in the lytic cycle of the parasite and with additional promising diagnostic value. However, the molecular identity of the antigens recognized remains to be elucidated.

Prognostic and predictive role of the PI3K-AKT-mTOR pathway in neuroendocrine neoplasms

Neuroendocrine neoplasms (NENs) are considered a heterogeneous and rare entity. Its natural history is influenced by multiple clinicopathological characteristics, which guide the management of these patients. The development of molecular biology reveals that the PI3K-AKT-mTOR pathway plays a relevant role in tumorigenesis and progression of NENs. Mammalian target of rapamycin (mTOR) inhibitors, targeted agents that block this pathway, has improved outcomes in neuroendocrine tumors (NETs). Different therapeutic approaches, such as somatostatin analogs, chemotherapy, peptide receptor radionuclide therapy, and targeted agents, have shown benefits in the treatment of NETs. However, there are not any established prognostic or predictive biomarkers to select the best therapy option to individualize treatment. Although a relation between alterations in the PI3K-AKT-mTOR pathway and clinical outcomes has not been found, these anomalies are considered attractive biomarkers. Additional molecular analysis should be integrated in future clinical trials' design to identify potential predictive or prognostic biomarkers

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Prognostic and predictive role of the PI3K-AKT-mTOR pathway in neuroendocrine neoplasms.

Neuroendocrine neoplasms (NENs) are considered a heterogeneous and rare entity. Its natural history is influenced by multiple clinicopathological characteristics, which guide the management of these patients. The development of molecular biology reveals that the PI3K-AKT-mTOR pathway plays a relevant role in tumorigenesis and progression of NENs. Mammalian target of rapamycin (mTOR) inhibitors, targeted agents that block this pathway, has improved outcomes in neuroendocrine tumors (NETs). Different therapeutic approaches, such as somatostatin analogs, chemotherapy, peptide receptor radionuclide therapy, and targeted agents, have shown benefits in the treatment of NETs. However, there are not any established prognostic or predictive biomarkers to select the best therapy option to individualize treatment. Although a relation between alterations in the PI3K-AKT-mTOR pathway and clinical outcomes has not been found, these anomalies are considered attractive biomarkers. Additional molecular analysis should be integrated in future clinical trials' design to identify potential predictive or prognostic biomarkers.

Integrative transcriptome and proteome analyses define marked differences between Neospora caninum isolates throughout the tachyzoite lytic cycle.

Neospora caninum is one of the main causes of transmissible abortion in cattle. Intraspecific variations in virulence have been widely shown among N. caninum isolates. However, the molecular basis governing such variability have not been elucidated to date. In this study label free LC-MS/MS was used to investigate proteome differences between the high virulence isolate Nc-Spain7 and the low virulence isolate Nc-Spain1H throughout the tachyzoite lytic cycle. The results showed greater differences in the abundance of proteins at invasion and egress with 77 and 62 proteins, respectively. During parasite replication, only 19 proteins were differentially abundant between isolates. The microneme protein repertoire involved in parasite invasion and egress was more abundant in the Nc-Spain1H isolate, which displays a lower invasion rate. Rhoptry and dense granule proteins, proteins related to metabolism and stress responses also showed differential abundances between isolates. Comparative RNA-Seq analyses during tachyzoite egress were also performed, revealing an expression profile of genes associated with the bradyzoite stage in the low virulence Nc-Spain1H isolate. The differences in proteome and RNA expression profiles between these two isolates reveal interesting insights into likely mechanisms involved in specific phenotypic traits and virulence in N. caninum. SIGNIFICANCE: The molecular basis that governs biological variability in N. caninum and the pathogenesis of neosporosis has not been well-established yet. This is the first study in which high throughput technology of LC-MS/MS and RNA-Seq is used to investigate differences in the proteome and transcriptome between two well-characterized isolates. Both isolates displayed different proteomes throughout the lytic cycle and the transcriptomes also showed marked variations but were inconsistent with the proteome results. However, both datasets identified a pre-bradyzoite status of the low virulence isolate Nc-Spain1H. This study reveals interesting insights into likely mechanisms involved in virulence in N. caninum and shed light on a subset of proteins that are potentially involved in the pathogenesis of this parasite.

Bruton's tyrosine kinase (BTK) as a promising target in solid tumors.

Bruton's tyrosine kinase (BTK) is a non-receptor intracellular kinase that belongs to the TEC-family tyrosine kinases together with bone marrow-expressed kinase (BMX), redundant-resting lymphocyte kinase (RLK), and IL-2 inducible T-Cell kinase (ITK). All these proteins play a key role in the intracellular signaling of both B and T lymphocytes. Recently, some preclinical data have demonstrated that BTK is present in certain tumor subtypes and in other relevant cells that are contributing to the tumor microenvironment such as dendritic cells, macrophages, myeloid derived suppressor cells and endothelial cells. Ibrutinib (PCI-32765) is an orally available small molecule that acts as an inhibitor of the BTK and is approved for the treatment of patients with some hematological malignancies. It has been suggested that ibrutinib may also have a potential antitumor activity in solid neoplasms. In this sense, ibrutinib has the ability to revert polarization of TCD4+ to Th1 lymphocytes to increase the cytotoxic ability of T CD8+ and to regulate tumor-induced immune tolerance by acting over tumor infiltrating cells activity and immunosuppressive cytokines release. Furthermore, based on its molecular activity and safety, ibrutinib has been considered as a partner for treatment combination with PI3K/AKT/mTOR inhibitors or with immune-checkpoint inhibitors, inhibiting immunosuppressive signals from the tumor microenvironment, and overcoming the immune resistance to current anti-PD1/PDL1 immunotherapeutic drugs by the CXCR4/CXCL2 pathway regulation. Currently, a broad range of different studies are evaluating the activity of ibrutinib either as single agent or in combination in patients with solid tumors.

Publisher's Note: Driven Open Quantum Systems and Floquet Stroboscopic Dynamics [Phys. Rev. Lett. 117, 250401 (2016)].

This corrects the article DOI: 10.1103/PhysRevLett.117.250401.

Systemic Besnoitiosis in a Juvenile Roe Deer (Capreolus capreolus).

Herein, we report the first incidence of systemic besnoitiosis in a male juvenile roe deer Capreolus capreolus. The animal was found dead in an area where bovine besnoitiosis is endemic and showed cachexia and multiple skin erosions in the metacarpal and metatarsal areas. Moreover, round and elevated white structures suggestive of Besnoitia spp. tissue cysts were also present. Twenty-eight tissue samples from different anatomical locations were collected for microscopic lesion and parasite detection through histopathology and PCR. Immunohistochemistry was performed to confirm Besnoitia-positive reaction in the tissue cysts. In addition, the identity of Besnoitia spp. in PCR-positive tissue samples was also investigated using microsatellite (MS) markers, and the comparison of protein disulphide isomerase gene sequences (BbPDI) of B. besnoiti and B. tarandi isolated from cattle and reindeer, respectively. Besnoitia cysts were detected in the skin (several parts), respiratory and upper digestive tracts, eyes, kidney, liver, testicle, cardiac muscle and lymphoid tissue. Remarkably, the presence of tissue cysts in the brain confirmed the capacity of Besnoitia spp. to form tissue cysts in the central nervous system (CNS). Finally, the Besnoitia species detected showed the same MS genotype as B. besnoiti, and BbPDI sequences from roe deer and two B. besnoiti isolates were genetically identical throughout multiple sequence alignment. Thus, for the first time, there is evidence that roe deer might act as an intermediate host of B. besnoiti. Further molecular analyses and parasite isolations are needed to corroborate these findings.

Driven Open Quantum Systems and Floquet Stroboscopic Dynamics.

We provide an analytic solution to the problem of system-bath dynamics under the effect of high-frequency driving that has applications in a large class of settings, such as driven-dissipative many-body systems. Our method relies on discrete symmetries of the system-bath Hamiltonian and provides the time evolution operator of the full system, including bath degrees of freedom, without weak-coupling or Markovian assumptions. An interpretation of the solution in terms of the stroboscopic evolution of a family of observables under the influence of an effective static Hamiltonian is proposed, which constitutes a flexible simulation procedure of nontrivial Hamiltonians. We instantiate the result with the study of the spin-boson model with time-dependent tunneling amplitude. We analyze the class of Hamiltonians that may be stroboscopically accessed for this example and illustrate the dynamics of system and bath degrees of freedom.

Vaccines for bovine neosporosis: current status and key aspects for development.

Bovine neosporosis is a worldwide concern due to its global distribution and great economic impact. Reproductive failure in cattle due to abortion leads to major economic losses associated with the disease. Currently, there is no treatment or vaccine available against abortion or transmission caused by Neospora caninum infection in cattle. However, vaccination is considered the best measure of control against bovine neosporosis. Several host and parasite factors can influence the dynamics of the infection in bovines. Moreover, the availability of well-defined infection models is a key factor for the evaluation of vaccine candidates. However, working with cattle is not easy due to difficult handling, facilities and costs, and therefore, 'more affordable' models could be used for screening of promising vaccines to establish proof of concept. So far, live-attenuated vaccines have shown good efficacy against exogenous transplacental transmission; however, they have relevant disadvantages and associated risks, which render inactivated or subunit vaccines the best way forward. The identification of novel potential targets and vaccines, and the application of innovative vaccine technologies in harmonized experimental animal models, will accelerate the development of an effective vaccine against bovine neosporosis.

Cell mediated immune responses in the placenta following challenge of vaccinated pregnant heifers with Neospora caninum.

The aim of the present study was to investigate and correlate the cell-mediated immune response and pathological changes at the maternal-fetal interface of Neospora-challenged pregnant cattle previously immunized with live and inactivated experimental vaccines. Pregnant heifers naïve to Neospora caninum were divided in 5 groups of 4 animals, each one immunized before mating: Group A heifers were intravenously (iv) immunized with 6.25 × 10(7) live tachyzoites of the NC-6 strain; group B heifers were immunized twice subcutaneously (sc) 3 weeks apart with native antigen extract of the NC-6 strain formulated with ISCOMs; group C heifers were sc immunized twice 3 weeks apart with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) of the NC-1 strain formulated with ISCOMs; group D heifers were sc injected with sterile phosphate-buffered saline (PBS) and group E heifers received sc ISCOM-matrix (ISCOMs without antigen). All groups were iv-challenged with 4.7 × 10(7) NC-1 tachyzoites at 70 days of gestation. Heifers were culled at day 104 of gestation and placentomes were examined to evaluate lesions and local cellular immune responses using histopathology, immunohistochemistry and real time-PCR. Immunohistochemistry was performed using bovine leucocyte specific antibodies. Cytokine expression and levels (IFN-γ, IL-4, IL-10, IL-12 and TNF-α) were measured using real-time reverse transcription-PCR and ELISA, respectively. Minimal inflammation was observed in group A placentomes; while placentomes from group B, C, D and E had moderate to severe infiltration with CD3(+), CD4(+), γδ-T cells, CD8(+) cells and macrophages being more numerous in groups B and E placentomes, when compared with groups C and D (P<0.001). Cytokine levels were significantly increased in the caruncles of animals of groups B and C in comparison with the other animal groups (P < 0.001). The results from this study showed that the strongest cellular immune responses were observed in the placentomes of animals that were immunized with inactivated vaccines (groups B and C) and in the placentomes of animals that were sc-sham-inoculated (groups D and E). On the other hand, animals that were immunized with live tachyzoites showed a milder immune cell infiltration to the placenta possibly due to the existence of a protective systemic maternal immune response that helped to minimize N. caninum infection at the maternal-fetal interface.

Proteomics reveals differences in protein abundance and highly similar antigenic profiles between Besnoitia besnoiti and Besnoitia tarandi.

Besnoitia besnoiti and Besnoitia tarandi are two cyst-forming apicomplexan parasites of the genus Besnoitia. B. besnoiti uses cattle as an intermediate host, in which it causes a disease that progresses in two sequential phases: the acute anasarca stage and the chronic scleroderma stage. Reindeer and caribou act as intermediate hosts for B. tarandi, which causes clinical signs similar to those caused by B. besnoiti. Previous studies demonstrated high molecular similarity, as determined by 18S and ITS-1 RNA sequences, between these Besnoitia spp., and strong serological cross-reactivity between these species has recently been demonstrated. Thus, a difference gel electrophoresis approach and mass spectrometry analysis were used to describe the proteomes and explore differences in protein abundance between B. besnoiti and B. tarandi in tachyzoite extracts. Immunoproteomes were also compared using 2-DE immunoblotting with polyclonal sera from experimentally infected rabbits. From approximately 1400 spots detected in DIGE-gels, 28 and 29 spots were differentially abundant in B. besnoiti and B. tarandi tachyzoites, respectively (± 1.5-fold, p<0.05). Four and 13 spots were exclusively detected in B. besnoiti and B. tarandi, respectively. Of the 32 differentially abundant spots analyzed by MALDI-TOF/MS, 6 up-regulated B. besnoiti proteins (LDH; HSP90; purine nucleoside phosphorylase and 3 hypothetical proteins) and 6 up-regulated B. tarandi proteins (G3PDH; LDH; PDI; mRNA decapping protein and 2 hypothetical proteins) were identified. Interestingly, no specific antigen spots were recognized by sera on any of the Besnoitia species studied and a similar antigen profile has been observed for B. tarandi and B. besnoiti sera when cross reactions were studied. This fact corroborates the difficulty in discerning Besnoitia infections using current serological assays. The present study underscores the importance of sequencing the B. besnoiti genome for species diversity studies of the genus Besnoitia.

Comparison of host cell invasion and proliferation among Neospora caninum isolates obtained from oocysts and from clinical cases of naturally infected dogs.

In a previous study we have shown that the in vitro invasion rate (IR) and tachyzoite yield (TY) are associated with the virulence phenotypes of Neospora caninum isolates of bovine origin. In addition, we recently observed marked differences in virulence when canine isolates were compared in a pregnant BALB/c mouse model. In this study, we investigated whether invasion and proliferation capacities could be used as virulence-related N. caninum phenotypic traits. Of the isolates compared in mice, four canine isolates obtained from oocysts (Nc-Ger2, Nc-Ger3, Nc-Ger-6, Nc-6 Arg) had shown a low-moderate virulence, and two further isolates obtained from dogs with neurological signs (Nc-Bahia, Nc-Liv) were highly virulent. The IR for each isolate was determined by a plaque assay and the counting of immunofluorescence-labeled parasitophorous vacuoles at 3 days post-inoculation (p.i.). The TY was determined by the quantification of tachyzoites at 56 h p.i. by real-time PCR. Most of the canine isolates showed similar IR values under controlled invasion conditions for 4h and 72 h p.i., indicating a limited time period for invasion similar to that observed for bovine isolates. The Nc-Ger3, Nc-Bahia, and Nc-Liv isolates showed a significantly higher IR and TY than the Nc-Ger2 and Nc-Ger6 isolates (P<0.0001). A correlation was found between the IRs and TY (ρ>0.885, P<0.033), as well as between the TY and both dam morbidity (ρ=0.8452, P<0.033) and pup mortality (ρ>0.8117, P<0.058) in mice. These results demonstrate the importance both the invasive and proliferative capacities have on the virulence of canine N. caninum isolates.

Neospora caninum tachyzoites inoculated by the conjunctival route are not vertically transmitted in pregnant cattle: a descriptive study.

The aim of this study was to evaluate whether Neospora caninum tachyzoites (Nc-1) inoculated by the conjunctival route in pregnant cows were able to generate infection in their fetuses. Group 1 contained 2 naturally infected cows; group 2 contained two cows inoculated intravenously with 2.5 × 10(8) tachyzoites, group 3 contained two cows inoculated with 2.5 × 10(8) tachyzoites by the conjunctival route, and group 4 contained two uninfected control cows. The four inoculated cows from groups 2 and 3 were challenged at 23 weeks of gestation. An indirect fluorescent antibody test (IFAT), recombinant NcGRA7-based ELISA, ELISA for IgG subisotypes and Western blot analysis were assessed to characterize the humoral immune response in dams. Sera from their fetuses were tested also using Western blot analysis. Routine microscopic evaluation of H&E stained fetal tissues was made and any fetal tissues and placentas with lesions compatible with Neospora-infection were processed by immunohistochemistry (IHC). DNA extraction from fresh and formalin-fixed, paraffin-embedded fetal tissues were tested by nested PCR. All dams from groups 1, 2 and 3 were seropositive by IFAT, rNcGRA7-based-ELISA and Western blot. IgG1/IgG2 ratios were ≤ 1 at weeks 27 and 29 of gestation. Only fetuses from groups 1 and 2 developed N. caninum specific antibodies by Western blot. Histopathological lesions compatible with those caused by N. caninum were observed in fetuses from groups 1 and 2. N. caninum cysts and tachyzoites were observed by IHC on fetal tissues from groups 1 and 2. Only fetal samples from group 2 were positive by PCR. Further work is needed not only to characterize the cellular immune response but also to clarify the consequences on the dam after conjunctival inoculation of N. caninum tachyzoites. This study shows that N. caninum tachyzoites inoculated by the conjunctival route were not vertically transmitted in pregnant cows.