Dynamics of anti-Toxoplasma gondii antibodies and seroconversion during pregnancy and lactation in naturally infected goats.

The objective of this work was to evaluate the dynamics of anti-T. gondii antibodies and seroconversion in naturally infected goats from the last third of pregnancy to 100 days of lactation and relate it to hematological and dehydration parameters. Blood samples were obtained from 56 goats in the different physiological states (pregnancy, kidding and lactation) as in different years (2019, 2021 and 2022). A total of 266 serum samples were obtained and evaluated by indirect fluorescent antibody test (IFAT) to end titer. The overall T. gondii seropositivity was 80.4% (45/56), with titers ranging from 100 to 25.600. The goats older than 3 years (4967 ± 1329) had significantly higher IFAT titers than the younger goats (2705 ± 681). The highest rate of positive seroconversion 31.1% (14/45) was found between kidding and 70 days of lactation; and of negative seroconversion 28.9% (13/45) between late pregnancy and kidding. The highest proportion of slightly dehydrated animals was found in the last third of pregnancy (14/25) and kidding (9/28). The correlation between seroconversion and T. gondii antibody titers was negative to the established dehydration index. These data suggest that in all physiological states and at different ages of goats, there is seroconversion which is not related to hydration status. Pregnancy, kidding and peak of lactation are stressful physiological periods, facilitating the reactivation of chronic T. gondii infections which are expressed by higher antibodies titers.

COVID-19-related premature ovarian insufficiency: case report and literature review.

OBJECTIVE: The aim of this study is to present the case report of a 36-year-old woman developing premature ovarian insufficiency (POI) after COVID-19 and review the literature referring to the possible impact of SARS-CoV-2 infection on female reproduction. METHODS: A 36-year-old nulligravida with normal menstrual cycles, non-smoker, with a normal body mass index and no pelvic surgery or oncological treatment in her medical history presented to the Infertility Center of the Institute of Mother and Child in Warsaw after a year of unsuccessful attempts to get pregnant. During diagnostic process she was affected by COVID-19 with a mild manifestation and thereafter she presented amenorrhea with intense hot flushes. Further diagnostic confirmed the diagnosis of POI. RESULTS: There is a strong molecular basis for a possible effect of SARS-CoV-2 infection on the female reproductive system; however, the results of available research are conflicting. All of these aspects are discussed in detail. CONCLUSIONS: SARS-CoV-2 infection may cause serious complications that cast a long shadow on a patient's future life and health. Further research is needed to assess the real impact of SARS-CoV-2 infection on female reproductive health, as well as potential preventive and therapeutic strategies for women affected with COVID-19.

Eleven years of Toxoplasma gondii serological follow-up in a goat herd and association of toxoplasmosis with reproductive losses.

Toxoplasmosis is considered one of the most important causes of abortion in small ruminants. The aim of this study was to evaluate the relationship between Toxoplasma gondii antibody titres and reproductive losses over an 11 year period in a goat farm located in Buenos Aires province, Argentina. Blood samples were obtained from 85 goats, representing three breeds, during the last third of gestation (n = 165 gestations), in consecutive pregnancies (2008-2019), and from 51 goats during kidding to analyze seroconversion. Serum was evaluated by IFAT with T. gondii antigen, using 1:100 dilution as the cut-off titre and processed to end titre. An overall reproductive loss of 31% (51/165) was detected, including 16.4% (27/165) abortions and 14.6% (24/165) perinatal deaths. The seropositivity to T. gondii was 100% (85/85) with all animals positive in successive samplings and, therefore, considered chronically infected. Antibody titres showed average values greater than 1100 in each year and breed group. Differences in antibody levels were associated with breed and were lower in those that were predominately Creole and higher in those that were predominately Saanen. Seroconversion was detected in 16.2% (6/37) and 57.1% (8/14) of goats from the Creole and Sannen breed groups, respectively. There were no significant differences in the antibody titre average between goats with reproductive losses and those with healthy kids, although the goats with perinatal deaths had a significantly higher titre average. These results suggest reinfection or reactivation, although no association with reproductive losses was observed. Higher antibody titres were associated with perinatal deaths. The high T. gondii antibody titres in a farm with 100% seroprevalence did not allow for association with reproductive losses, particularly abortion, to be assessed.

The power of the Mediator complex-Expanding the genetic architecture and phenotypic spectrum of MED12-related disorders.

MED12 is a member of the large Mediator complex that controls cell growth, development, and differentiation. Mutations in MED12 disrupt neuronal gene expression and lead to at least three distinct X-linked intellectual disability syndromes (FG, Lujan-Fryns, and Ohdo). Here, we describe six families with missense variants in MED12 (p.(Arg815Gln), p.(Val954Gly), p.(Glu1091Lys), p.(Arg1295Cys), p.(Pro1371Ser), and p.(Arg1148His), the latter being first reported in affected females) associated with a continuum of symptoms rather than distinct syndromes. The variants expanded the genetic architecture and phenotypic spectrum of MED12-related disorders. New clinical symptoms included brachycephaly, anteverted nares, bulbous nasal tip, prognathism, deep set eyes, and single palmar crease. We showed that MED12 variants, initially implicated in X-linked recessive disorders in males, may predict a potential risk for phenotypic expression in females, with no correlation of the X chromosome inactivation pattern in blood cells. Molecular modeling (Yasara Structure) performed to model the functional effects of the variants strongly supported the pathogenic character of the variants examined. We showed that molecular modeling is a useful method for in silico testing of the potential functional effects of MED12 variants and thus can be a valuable addition to the interpretation of the clinical and genetic findings.

Pampas fox (Lycalopex gymnocercus) new intermediate host of Sarcocystis svanai (Apicomplexa: Sarcocystidae).

Several Sarcocystis spp. have carnivores as definitive host and sarcocysts are common in muscles of herbivores (intermediate host). However, sarcocysts have been found in muscles of wild and domestic carnivores suggesting they are intermediate host for some Sarcocystis spp. Here, we report mature sarcocysts in the muscles of Pampas fox (Lycalopex gymnocercus). A total of 36 free-living foxes were analyzed. Different skeletal muscles were assessed by microscopic and molecular methods. Cysts and/or DNA of Sarcocystis sp. were detected in 61.1% (22/36) foxes. Histopathology revealed the presence of sarcocysts in 52.8% (19/36) foxes. The tongue and masseter were the muscles more frequently infected. Of all the samples processed by homogenization of pooled muscles of each animal, 45.4% (10/22) evidenced muscle cysts and 68.2% (15/22) resulted positives by PCR. Individual cysts obtained from the ten positive samples in direct microscopic examination were all positive by PCR. Five amplicons from individual cysts from different samples were selected for sequencing together with four PCR products obtained from the pooled muscles. All nine sequences shared a high identity among them (99.8-100%) and showed the highest identity by BLAST (99%) with a S. svanai sequence (KM362428) from a North American dog. By transmission electron microscopy, the sarcocyst wall was thin (<1µm), had minute undulations, with tiny evaginations and without evident villar protrusions. The cyst wall type is referred as "type 1". Sarcocystis svanai infects L. gymnocercus with a high prevalence and the presence of mature sarcocysts suggests the role of the Pampas fox as natural intermediate host. The definitive host of S. svanai remains unknown.

Sarcocystis masoni, n. sp. (Apicomplexa: Sarcocystidae), and redescription of Sarcocystis aucheniae from llama (Lama glama), guanaco (Lama guanicoe) and alpaca (Vicugna pacos).

There is considerable confusion concerning the species of Sarcocystis in South American camelids (SAC). Several species names have been used; however, proper descriptions are lacking. In the present paper, we redescribe the macroscopic sarcocyst forming Sarcocystis aucheniae and describe and propose a new name, Sarcocystis masoni for the microscopic sarcocyst forming species. Muscles samples were obtained from llamas (Lama glama) and guanacos (Lama guanicoe) from Argentina and from alpacas (Vicugna pacos) and llamas from Peru. Individual sarcocysts were processed by optical and electron microscopy, and molecular studies. Microscopic sarcocysts of S. masoni were up to 800 µm long and 35-95 µm wide, the sarcocyst wall was 2·5-3·5 µm thick, and had conical to cylindrical villar protrusions (vp) with several microtubules. Each vp had 11 or more rows of knob-like projections. Seven 18S rRNA gene sequences obtained from sarcocysts revealed 95-96% identity with other Sarcocystis spp. sequences reported in the GenBank. Sarcocysts of S. aucheniae were macroscopic, up to 1·2 cm long and surrounded by a dense and laminar 50 µm thick secondary cyst wall. The sarcocyst wall was up to 10 µm thick, and had branched vp, appearing like cauliflower. Comparison of the 11 sequences obtained from individual macroscopic cysts evidenced a 98-99% of sequence homology with other S. aucheniae sequences. In conclusion, 2 morphologically and molecularly different Sarcocystis species, S. masoni (microscopic cysts) and S. aucheniae (macroscopic cysts), were identified affecting different SAC from Argentina and Peru.

Toxoplasma gondii and Neospora caninum infections in goat abortions from Argentina.

The aims of this study were to identify the occurrence of Toxoplasma gondii and Neospora caninum abortions in goats from Argentina by serological, macroscopical and microscopical examination and bioassay, and to characterize the obtained isolates by molecular techniques. For this purpose, 25 caprine fetal fluids, 18 caprine fetal brains and 10 caprine placentas from 8 dairy/meat goat farms from Argentina were analyzed. Gestational age of the aborted fetuses was determined in 18 cases. Protozoal infections were detected by at least one of the applied diagnostic techniques in 44% (11/25) of examined fetuses; specifically, 24% (6/25) were positive to T. gondii, 8% (2/25) were positive to N. caninum and 12% (3/25) were positive to both parasites. In this study IFAT titers were similarly distributed in younger and older fetuses. Macroscopical and microscopical examination of one placenta revealed chalky nodules in the fetal cotyledons and normal intercotyledonary areas, as well as necrosis and calcification of mesenchymal cells in villi. Tachyzoites were observed in peritoneal wash from 2 mice inoculated with brain and a pool of brain and placenta of two fetuses. Cell culture growth of tachyzoites was achieved from one inoculated mouse, and confirmed as T. gondii by PCR. The T. gondii isolate was identified as atypical or non-canonical by nested-PCR-RFLP. This is the first study that investigated the involvement of N. caninum and T. gondii in cases of goat abortion in Argentina.

Neospora caninum NC-6 Argentina induces fetopathy in both serologically positive and negative experimentally inoculated pregnant dams.

Neospora caninum infection is a major cause of abortion in cattle. The objectives of this study were to genetically characterize the N. caninum NC-6 Argentina isolate using a multilocus microsatellite analysis approach and to study its biological behavior by experimental inoculations into seronegative and seropositive pregnant cattle, evaluating the humoral and cellular immune response elicited and the occurrence of transplacental transmission and fetopathy. Pregnant cows (65 days of gestation) seropositive and seronegative to N. caninum were intravenously inoculated with tachyzoites of the NC-6 Argentina N. caninum strain and slaughtered at 108 ± 2 days of gestation. Serum samples were analyzed for N. caninum antibodies by indirect fluorescent antibody test. The cellular immune response was analyzed by detection of gamma interferon (γIFN) production in blood cells. Tissue samples from dams, fetuses, and placental cotyledons were processed by histopathological and immunohistochemical techniques and examined for N. caninum DNA by PCR. Positive DNA samples were further analyzed by multilocus microsatellite typing for N. caninum. Inoculated animals had significantly higher N. caninum antibody titers and γIFN production than control animals. One seropositive inoculated cow aborted, one seronegative cow had a non-viable fetus, and the remaining fetuses from the experimentally inoculated dams had histopathologic lesions. The PCR was positive in 3/4 fetuses from seronegative inoculated cows and in 2/3 fetuses from seropositive inoculated cows. Multilocus microsatellite analysis revealed that the N. caninum DNA present in fetuses and placentas had an identical pattern to NC-6 Argentina strain. The NC-6 Argentina strain proved to be able to cross the placenta and to induce fetopathy in both the seropositive and seronegative dams.

TrkB expression level correlates with metastatic properties of L1 mouse sarcoma cells cultured in non-adhesive conditions.

OBJECTIVES: Ability of a cell to survive without adhesion, and to overcome anoikis, is indispensable for malignant cell invasion and metastasis formation. It has previously been shown that TrkB -neutrophin growth factor receptor might be involved in suppression of apoptosis, induced by the lack of adhesion. The aim of our study was to analyse changes in expression of genes and proteins as well as in biological properties of cancer cells cultured without adhesion. A mouse sarcoma, stable, adherent L1 cell line, derived from a spontaneously arisen Balb/c mouse lung tumour, was established in vitro. MATERIALS AND METHODS: L1 cells resistant to anoikis were established by culture of L1 cells without adhesion, followed by selection of clones with elevated expression levels of TrkB protein. Biological characteristics of the cells were studied by migration/invasion tests and colony forming assay. Gene expression analysis was performed by with the aid of cDNA Gene Expression Array and Real-Time PCR. In vivo experiments were conducted in syngeneic Balb/c mice. RESULTS: Significant changes in gene expression, including higher expression level of TrkB, were found in cells that were able to survive without adhesion. Selected TrkB-expressing clones were found to have higher clonogenicity and invasive potential, formed more colonies in mouse lungs, and induced larger tumours, when injected subcutaneously into Balb/c mice. CONCLUSION: Lack of adhesion induced significant changes in the cancer cells' behaviour, which may result from alterations in gene and protein expression levels, including changes in anoikis-connected protein - TrkB.

Mouse sarcoma L1 cell line holoclones have a stemness signature.

Accumulating data suggest that cancers contain a fraction of cells called cancer stem cells (CSCs), that may be responsible for upkeep and relapses of disease. In experimental settings, CSCs are regarded as most effective at tumour initiation in in vivo assays. Since the first isolation of cancer stem cells from acute myeloid leukaemia in 1994, cancer stem cells have been identified in human solid tumours and they have also been found in the established cell lines, based on ability of CSCs to form in vitro colonies of a specific morphology, called holoclones. Our study examined the ability of a mouse sarcoma cell line, derived from a lung metastasis of a BALB/c mouse and established as a stably growing line (L1), to produce holoclones in vitro. We aimed to verify a stemness signature of the holoclone cells. The L1 cell line was found to form holoclone colonies in vitro, which were shown to contain a percentage of CSC-like cells. A fraction of the L1 cells was able to repopulate the original cell line, and presented an increased clonogenic and metastatic potential (18th passage). In addition, MTT assay and flow cytometry of the side population fraction revealed that these cells were more resistant to chemotherapeutic drugs than the original cell line, and over-expressed the anti-apoptotic genes, GRP78 and GADD153. We conclude that mouse L1 sarcoma cell line contains CSC-like cells.

Effect of transfection with a gene coding for the fibronectin FNIII/10 fragment upon contact inhibition of C3H10T1/2 fibroblasts.

The density-dependent growth inhibition of non-transformed cells may be associated with inefficient transduction of the proliferative signal from cell adhesion molecules. To verify this concept, the C3H10T1/2 fibroblasts were stably transfected with the gene coding for the fibronectin fragment III/10 (FNIII/10). This resulted in differences in gene's expression between original C3H10T1/2 cells and their FNIII/10 transfectants. No significant differences in growth properties were observed in the original or in the transfected cells. C3H10T1/2 cells and their transfectants, when co-cultured, displayed more cells at confluence than the cells cultured alone. Moreover, co-cultured C3H10T1/2 cells and their transfectants showed elevated levels of phospho-ERK1/2 compared to homogenous cultures. Results obtained indicate that cellular homogeneity is responsible for density-dependent growth inhibition.

Cellular quiescence induced by contact inhibition or serum withdrawal in C3H10T1/2 cells.

Either confluence or serum withdrawal may cause growth arrest of cultured non-transformed cells. Here, we compared sparsely populated and confluent C3H10T1/2 cells with and without serum-containing medium. The following proliferation-relevant end points were examined: cell-cycle distribution, Ki-67 antigen presence, the level of the von Hippel-Lindau (VHL) protein, and gene expression, determined using a microarray approach. In sparse/logarithmic cultures, the fraction of cells in G(0)/G(1) phase increased from 55 to 85% following serum withdrawal. Moreover, the fraction of Ki-67 positive cells dropped from 89 to 47%. In confluent cultures, the majority of cells (80%) were in G(0)/G(1) phase and only 25-30% were Ki-67 positive, regardless of serum presence. In both serum-deprived and contact-inhibited cultures, significant and distinct changes in gene expression were observed. Serum deprivation of sparsely cultured cells resulted in significant over-expression of several transcription factors, while confluent cells showed elevated expression of genes coding for Wnt6, uPar, Tdag51, Egr1, Ini1a and Mor1. These results indicate that contact inhibition and serum withdrawal lead to cellular quiescence through distinct genetic and molecular mechanisms.