An IgG1 single-dilution avidity ELISA predicts cross-protection against heterologous foot-and-mouth disease virus challenge after vaccination.

This study aims to analyze if the results from different serological assays, used alone or combined, could match the outcome of challenge infection with foot-and-mouth disease virus (FMDV) after vaccination in cattle. Day-of-challenge sera from animals that had been vaccinated 21 days before with monovalent formulations containing inactivated A Iran 96 or A Iran 99 virus strains were used. Challenge and serology were performed with A22 Iraq strain. IgG1 titers and total-IgG avidity indexes were significantly higher in protected animals (p < 0.01) while IgG2-titers were not related to protection (p > 0.05). An IgG1 avidity ELISA was developed to analyze in one step, IgG1 levels and avidity. This assay estimated protection with 96 % accuracy. A strong agreement with challenge results was achieved (K = 0.85), suggesting a role of high-affinity IgG1 in protection against FMDV. These results support the assessment of the single dilution IgG1-Avidity ELISA to predict cross-protection in FMDV-vaccinated cattle.

Enhanced infectivity of bovine viral diarrhoea virus (BVDV) in arginase-producing bovine monocyte-derived macrophages.

Macrophages are important cells of the innate immunity that play a major role in Bovine Viral Diarrhea Virus (BVDV) pathogenesis. Macrophages are not a homogenous population; they exist in different phenotypes, typically divided into two main categories: classically (pro-inflammatory) and alternatively activated (anti-inflammatory) or M1 and M2, respectively. The role of bovine macrophage phenotypes on BVDV infection is still unclear. This study characterized the interaction between BVDV, and monocyte-derived macrophages (Mo-Mφ) collected from healthy cattle and polarized to an M1 or M2 state by using LPS, INF-γ, IL-4 or azithromycin. Arginase activity quantitation was utilized as a marker of the M2 Mo-Mφ spectrum. There was a significant association between arginase activity and the replication rate of BVDV strains of different genotypes and biotypes. Inhibition of arginase activity also reduced BVDV infectivity. Calves treated with azithromycin induced Mo-Mφ of the M2 state produced high levels of arginase. Interestingly, azithromycin administered in vivo increased the susceptibility of macrophages to BVDV infection ex vivo. Mo-Mφ from pregnant dams and calves produced higher arginase levels than those from non-pregnant adult animals. The increased infection of arginase-producing alternatively activated bovine macrophages with BVDV supports the need to delve into a possible leading role of M2 macrophages in establishing the immune-suppressive state during BVDV convalescence.

Serological screening of SARS-CoV-2 infection in companion animals of Buenos Aires suburbs.

The Coronavirus Disease 2019 (COVID-19) is a zoonotic disease caused by the pandemic virus SARS-CoV-2. Domestic and wild animals are susceptible to infection and are potential reservoirs for virus variants. To date, there is no information about the exposure of companion animals in Buenos Aires Suburbs, the area with the largest population in Argentina where the highest number of COVID-19 human cases occurred during the first infection wave. Here we developed a multi-species indirect ELISA to measure antibodies reactive to the SARS-CoV-2 receptor-binding domain (RBD) from several vertebrates constituting the class Mammalia, making it a valuable tool for field serosurveillance. The ELISA cut-off value was estimated by sera from dogs, cats, cattle, and pigs sampled before 2019 (n = 170), considering a 98% percentile and a grey zone to completely exclude any false positive result. Specificity was confirmed by measuring levels of neutralizing antibodies against canine coronavirus, the avidity of specific antibodies, and their capacity to impede the binding of a recombinant RBD protein to VERO cells in an In-Cell ELISA. Sera from 464 cats and dogs sampled in 2020 and 2021 ("pandemic" samples) were assessed using the RBD-ELISA. Information on COVID-19 disease in the household and the animals' lifestyles was collected. In Buenos Aires Suburbs cats were infected at a higher proportion than dogs, seroprevalence was 7.1 and 1.68%, respectively. Confirmed COVID-19 in the caregivers and outdoor lifestyle were statistically associated with seropositivity in cats. The risk of cats getting infected living indoors in COVID-19-negative households was null. The susceptibility of mammals to SARS-CoV-2, the possibility of transmission between animals themselves and humans, together with the free-roaming lifestyle typical of Buenos Aires suburban companion animals, urge pursuing responsible animal care and avoiding human interaction with animals during the disease course. The multi-species RBD-ELISA we developed can be used as a tool for serosurveillance of SARS-CoV-2 infection in mammalians (domestic and wild), guiding further targeted virological analyses to encounter susceptible species, interspecies transmission, and potential virus reservoirs in our region.

Prevalencia sérica de Neospora caninum y factores de riesgo asociados a su transmisión en tambos de la provincia de Salta, Argentina / Seroprevalence and risk factors associated with Neospora caninum in dairy farms from the Province of Salta, Argentina

Resumen El objetivo de este trabajo fue conocer la seroprevalencia de Neospora caninum en los bovinos de los tambos del Valle del Lerma (Salta, Argentina) y los factores de riesgo asociados a la transmisión de este parásito en esta región. Se tomaron muestras de suero de aproximadamente 40 vacas en cada tambo, que fueron analizadas por ELISA indirecto para detectar anticuerpos contra N. caninum. También se discriminó entre infecciones crónicas y agudas midiendo la avidez de dichos anticuerpos. Todos los tambos presentaron al menos un bovino seropositivoy la media fue de 35,3 ± 14,9% de animales positivos. También se detectaron anticuerpos específicos en caninos presentes en 9 de los 16 tambos, con un valor de seropositi- vidad del 71,7 ± 19,9%. El 56,3% de los bovinos seropositivos cursaban infecciones agudas. Se halló una asociación negativa entre la seroprevalencia y el índice de avidez de los anticuerpos específicos, lo que indica que la presencia de animales con infecciones agudas se asocia a mayor seroprevalencia. Los campos con pastoreo presentaron mayor cantidad de infecciones recien tes. Estos resultados revelan por primera vez la importancia de este parásito en los tambos de la región y la necesidad de propiciar el desarrollo de programas de control considerando los distintos factores de riesgo que afectan la situación epidemiológica de la enfermedad.

Abstract The objective of this work was to determine the seroprevalence of Neospora caninum in cattle in Valle de Lerma, province of Salta, Argentina, and the risk factors associated with the disease. Serum samples were taken from 40 cows in each dairy herd, which were analyzed by indirect ELISA to detect antibodies against N. caninum. Chronic and acute infections were discriminated by measuring the avidity of these antibodies. All the herds exhibited at least one seropositive animal, the mean being 35.3 ± 14.9% of positive animals. Specific antibodies were also detected in dogs present in 9of the herds, which showed a seropositivity value of 71.7% ± 19.9%. Among the seropositive animals, 56.3% showed acute infections. A negative association was found between seroprevalence and the avidity index of specific antibodies, indicating that the presence of animals with acute infections is associated with higher seroprevalence. Fields with grazing showed more recent infections. These results show for the first time the importance of this parasite in this particular region and the need to promote the development of control programs considering the different risk factors that affect the epidemiological situation of the disease.

Review of antiviral peptides for use against zoonotic and selected non-zoonotic viruses.

Viruses remain one of the leading causes of animal and human disease. Some animal viral infections spread sporadically to human populations, posing a serious health risk. Particularly the emerging viral zoonotic diseases such as the novel, zoonotic coronavirus represent an actual challenge for the scientific and medical community. Besides human health risks, some animal viral infections, although still not zoonotic, represent important economic loses to the livestock industry. Viral infections pose a genuine concern for which there has been an increasing interest for new antiviral molecules. Among these novel compounds, antiviral peptides have been proposed as promising therapeutic options, not only for the growing body of evidence showing hopeful results but also due to the many adverse effects of chemical-based drugs. Here we review the current progress, key targets and considerations for the development of antiviral peptides (AVPs). The review summarizes the state of the art of the AVPs tested in zoonotic (coronaviruses, Rift Valley fever viruses, Eastern Equine Encephalitis Virus, Dengue and Junín virus) and also non-zoonotic farm animal viruses (avian and cattle viruses). Their molecular target, amino acid sequence and mechanism of action are summarized and reviewed. Antiviral peptides are currently on the cutting edge since they have been reported to display anti-coronavirus activity. Particularly, the review will discuss the specific mode of action of AVPs that specifically inhibit the fusion of viral and host-cell membranes for SARS-CoV-2, showing in detail some important features of the fusion inhibiting peptides that target the spike protein of these risky viruses.

[Seroprevalence and risk factors associated with Neospora caninum in dairy farms from the Province of Salta, Argentina]. / Prevalencia sérica de Neospora caninum y factores de riesgo asociados a su transmisión en tambos de la provincia de Salta, Argentina.

The objective of this work was to determine the seroprevalence of Neospora caninum in cattle in Valle de Lerma, province of Salta, Argentina, and the risk factors associated with the disease. Serum samples were taken from 40 cows in each dairy herd, which were analyzed by indirect ELISA to detect antibodies against N. caninum. Chronic and acute infections were discriminated by measuring the avidity of these antibodies. All the herds exhibited at least one seropositive animal, the mean being 35.3 ± 14.9% of positive animals. Specific antibodies were also detected in dogs present in 9of the herds, which showed a seropositivity value of 71.7% ± 19.9%. Among the seropositive animals, 56.3% showed acute infections. A negative association was found between seroprevalence and the avidity index of specific antibodies, indicating that the presence of animals with acute infections is associated with higher seroprevalence. Fields with grazing showed more recent infections. These results show for the first time the importance of this parasite in this particular region and the need to promote the development of control programs considering the different risk factors that affect the epidemiological situation of the disease.

Interferon lambda protects cattle against bovine viral diarrhea virus infection.

Interferon lambda (IFN-λ) plays an important role in inducing an antiviral state in mucosal surfaces and has been used as an effective biotherapeutic against several viral diseases. Here we performed a proof of concept study on the activity of a biologically active recombinant bovine IFN-λ (rIFN-λ) produced in eukaryotic cells against Bovine Viral Diarrhea Virus (BVDV) in cattle. We first confirmed the lack of toxicity of different concentrations of rIFN-λ in bovine peripheral blood cells and the safety of its subcutaneous application in calves in doses up to 12 IU/kg. The antiviral activity of the rIFN-λ against BVDV was assessed in calves that were inoculated with 6 IU/kg of rIFN-λ (n = 4) or mock-treated (n = 2) two days before and after challenge with a BVDV type-2 non-cytopathic strain. Mock-treated animals developed respiratory disease, shedded the virus from 4 to 7 days post-infection (dpi) and had viremia between 4 and 14 dpi. Conversely, calves treated with rIFN-λ did not develop clinical symptoms. The virus was not found in nasal secretions or sera. Only one animal had a positive viral RNA detection in serum at 7 dpi. All infected animals treated with rIFN-λ increased systemic type-I IFNs levels at 4 dpi. The antiviral treatment induced an earlier onset of the anti-BVDV neutralizing antibodies. Altogether, these results constitute the proof-of-principle of bovine IFN-λ as an antiviral biotherapeutic to protect cattle against the clinical disease caused by BVDV.

Bovine Interferon Lambda Is a Potent Antiviral Against SARS-CoV-2 Infection in vitro.

Interferon lambda (IFN-λ) is an antiviral naturally produced in response to viral infections, with activity on cells of epithelial origin and located in the mucosal surfaces. This localized activity results in reduced toxicity compared to type I IFNs, whose receptors are ubiquitously expressed. IFN-λ has been effective in the therapy of respiratory viral infections, playing a crucial role in potentiating adaptive immune responses that initiate at mucosal surfaces. Human IFN-λ has polymorphisms that may cause differences in the interaction with the specific receptor in the human population. Interestingly, bovine IFN-λ3 has an in silico-predicted higher affinity for the human receptor than its human counterparts, with high identity with different human IFN-λ variants, making it a suitable antiviral therapeutic candidate for human health. Here, we demonstrate that a recombinant bovine IFN-λ (rbIFN-λ) produced in HEK-293 cells is effective in preventing SARS-CoV-2 infection of VERO cells, with an inhibitory concentration 50% (IC50) between 30 and 50 times lower than that of human type I IFN tested here (α2b and ß1a). We also demonstrated the absence of toxicity of rbIFN-λ in human PBMCs and the lack of proinflammatory activity on these cells. Altogether, our results show that rbIFN-λ is as an effective antiviral potentially suitable for COVID-19 therapy. Among other potential applications, rbIFN-λ could be useful to preclude virus dispersion to the lungs and/or to reduce transmission from infected people. Moreover, and due to the non-specific activity of this IFN, it can be potentially effective against other respiratory viruses that may be circulating together with SARS-CoV-2.

In-vivo Activity of IFN-λ and IFN-α Against Bovine-Viral-Diarrhea Virus in a Mouse Model.

Bovine-viral-diarrhea virus (BVDV) can cause significant economic losses in livestock. The disease is controlled with vaccination and bovines are susceptible until vaccine immunity develops and may remain vulnerable if a persistently infected animal is left on the farm; therefore, an antiviral agent that reduces virus infectivity can be a useful tool in control programs. Although many compounds with promising in-vitro efficacy have been identified, the lack of laboratory-animal models limited their potential for further clinical development. Recently, we described the activity of type I and III interferons, IFN-α and IFN-λ respectively, against several BVDV strains in-vitro. In this study, we analyzed the in-vivo efficacy of both IFNs using a BALB/c-mouse model. Mice infected with two type-2 BVDV field strains developed a viremia with different kinetics, depending on the infecting strain's virulence, that persisted for 56 days post-infection (dpi). Mice infected with the low-virulence strain elicited high systemic TNF-α levels at 2 dpi. IFNs were first applied subcutaneously 1 day before or after infection. The two IFNs reduced viremia with different kinetics, depending on whether either one was applied before or after infection. In a second experiment, we increased the number of applications of both IFNs. All the treatments reduced viremia compared to untreated mice. The application of IFN-λ pre- and post-infection reduced viremia over time. This study is the first proof of the concept of the antiviral potency of IFN-λ against BVDV in-vivo, thus encouraging further trails for a potential use of this cytokine in cattle.

A direct high-throughput in Cell-ELISA for measuring infectivity of cytopathic and non-cytopathic bovine viral diarrhoea virus strains applied to the assessment of antiviral activity.

Low-cost high-throughput methods applicable to any virus strain are required for screening antiviral compounds against multiple field strains. Colorimetric cell-viability assays are used for this purpose as long as the viruses are cytopathic (CP) in cell culture. However, bovine viral diarrhoea virus (BVDV) strains circulating in the field are mostly non-cytopathic (NCP). An In Cell-ELISA aimed to measure viral infectivity by detecting a conserved protein produced during viral replication (non-structural protein 3, "NS3") was developed. The ELISA is performed without harvesting the cells, directly on the 96-wells culture plate. NS3 In Cell-ELISA was tested for its ability to assess BVDV-specific antiviral activity of recombinant bovine type I and III IFNs. Results correlated to those measured by qRT-PCR and virus titration. NS3 In Cell-ELISA was also efficient in estimating the IC50 of two compounds with different antiviral activity. Estimation of the 50% inhibition dose of each IFN using six BVDV strains of different biotype and genotype showed that CP strains were more susceptible to both IFNs than NCP, while type 2 NCP viruses were more sensitive to IFN-I. The In Cell-ELISA format using a detector antibody against a conserved non-structural protein can be potentially applied to accurately measure infectivity of any viral strain.

In Vitro and In Vivo Characterization of a Typical and a High Pathogenic Bovine Viral Diarrhea Virus Type II Strains.

Non-cytopathic (ncp) type 2 bovine viral diarrhea virus (BVDV-2) is widely prevalent in Argentina causing high mortality rates in cattle herds. In this study, we characterized an Argentinean ncp BVDV-2 field isolate (98-124) compared to a high-virulence reference strain (NY-93), using in silico analysis, in vitro assays, and in vivo infections of colostrum-deprived calves (CDC) to compare pathogenic characters and virulence. In vitro infection of bovine peripheral blood mononuclear cells (PBMC) with BVDV 98-124 induced necrosis shortly after infection while NY-93 strain increased the apoptotic rate in infected cells. Experimental infection of CDC (n = 4 each) with these strains caused an enteric syndrome. High pyrexia was detected in both groups. Viremia and shedding were more prolonged in the CDC infected with the NY-93 strain. In addition, NY-93 infection elicited a severe lymphopenia that lasted for 14 days, whereas 98-124 strain reduced the leukocyte counts for 5 days. All infected animals had a diminished lymphoproliferation activity in response to a mitogen. Neutralizing and anti-NS3 antibodies were detected 3 weeks after infection in all infected calves. Virulence was associated with a more severe clinical score, prolonged immune-suppression, and a greater window for transmission. Studies of apoptosis/necrosis performed after in vitro PBMC infection also revealed differences between both strains that might be correlated to the in vivo pathogenesis. Our results identified 98-124 as a low-virulence strain.

Bovine Viral Diarrhea Virus Infects Monocyte-Derived Bovine Dendritic Cells by an E2-Glycoprotein-Mediated Mechanism and Transiently Impairs Antigen Presentation.

Infection of professional antigen presenting cells by viruses can have a marked effect on these cells and important consequences for the generation of subsequent immune responses. In this study, we demonstrate that different strains of bovine viral diarrhea virus (BVDV) infect bovine dendritic cells differentiated from nonadherent peripheral monocytes (moDCs). BVDV did not cause apoptosis in these cells. Infection of moDC was prevented by incubating the virus with anti-E2 antibodies or by pretreating the cells with recombinant E2 protein before BVDV contact, suggesting that BVDV infects moDC through an E2-mediated mechanism. Virus entry was not reduced by incubating moDC with Mannan or ethylenediaminetetraacetic acid (EDTA) before infection, suggesting that Ca(2+) and mannose receptor-dependent pathways are not mediating BVDV entry to moDC. Infected moDC did not completely upregulate maturation surface markers. Infection, but not treatment with inactivated virus, prevented moDC to present a third-party antigen to primed CD4(+) T cells within the first 24 hours postinfection (hpi). Antigen-presenting capacity was recovered when viral replication diminished at 48 hpi, suggesting that active infection may interfere with moDC maturation. Altogether, our results suggest an important role of infected DCs in BVDV-induced immunopathogenesis.

Immunization with Neospora caninum profilin induces limited protection and a regulatory T-cell response in mice.

Profilins are actin-binding proteins that regulate the polymerization of actin filaments. In apicomplexan parasites, they are essential for invasion. Profilins also trigger the immune response of the host by activating TLRs on dendritic cells (DCs), inducing the production of pro-inflammatory cytokines. In this study we characterized for the first time the immune response and protection elicited by a vaccine based on Neospora caninum profilin in mice. Groups of eight BALB/c mice received either two doses of a recombinant N. caninum profilin expressed in Escherichia coli. (rNcPRO) or PBS, both formulated with an aqueous soy-based adjuvant enriched in TLR-agonists. Specific anti-profilin antibodies were detected in rNcPRO-vaccinated animals, mainly IgM and IgG3, which were consumed after infection. Splenocytes from rNcPRO-immunized animals proliferated after an in vitro stimulation with rNcPRO before and after challenge. An impairment of the cellular response was observed in NcPRO vaccinated and infected mice following an in vitro stimulation with native antigens of N. caninum, related to an increase in the percentage of CD4+CD25+FoxP3+. Two out of five rNcPRO-vaccinated challenged mice were protected; they were negative for parasite DNA in the brain and showed no histopathological lesions, which were found in all PBS-vaccinated animals. As a whole, our results provide evidence of a regulatory response elicited by immunization with rNcPRO, and suggest a role of profilin in the modulation and/or evasion of immune responses against N. caninum.

Safety and immunogenicity of a soluble native Neospora caninum tachyzoite-extract vaccine formulated with a soy lecithin/ß-glucan adjuvant in pregnant cattle.

The global economic impact of Neospora caninum infection in cattle herds has promoted the development of vaccines that can be safely used during pregnancy. The aim of this study was to evaluate the safety and immunogenicity of a vaccine formulated with the soluble fraction of tachyzoite's lysate and a soy-based aqueous adjuvant (sNcAg/AVEC), which was protective in the mouse model and induced strong IFN-γ responses and high avidity antibodies in non-pregnant cattle. Ten pregnant heifers were vaccinated twice during the first trimester of gestation and 8 remained unvaccinated. Anti-N. caninum immune responses were efficiently primed by vaccination, evidenced by a quick induction of IgM serum titers (7dpv) and a prompt switch to high avidity IgG shortly after infection (performed at 78 or 225 days of gestation; n=5 each); while naïve cattle elicited lower IgG titers, with a delayed kinetics. High systemic IFN-γ levels were induced after infection which did not interfere with pregnancy. No local or systemic adverse effects were recorded along the study. Calves were born in term and in good health conditions, showing that the sNcAg/AVEC vaccine was safe when applied to healthy heifers during the first trimester of gestation.

Foot-and-mouth disease vaccination induces cross-reactive IFN-γ responses in cattle that are dependent on the integrity of the 140S particles.

Interferon-γ (IFN-γ) recall responses against foot-and-mouth disease virus (FMDV) in FMD vaccinated cattle are utilized to study T-lymphocyte immunity against this virus. Here, a recall IFN-γ assay based on a commercial ELISA was set up using 308 samples from naïve and vaccinated cattle. The assay was used to study cross-reactive responses between different FMDV vaccine strains. Blood samples from cattle immunized with monovalent vaccines containing A24/Cruzeiro/Brazil/55, A/Argentina/2001 or O1/Campos/Brazil/58 strains were tested using purified-inactivated FMDV from homologous and heterologous strains. A24/Cruzeiro was the most efficient IFN-γ inducer in all vaccinated animals, both when included in the vaccine or as stimulating antigen. We demonstrate that this was mainly due to the structural stability of the whole viral particle. These results show that IFN-γ production relies on the presence of 140S particles that can maintain their integrity along the incubation process in vitro, and throughout the vaccine's shelf-life, when used in vivo.

Low dose of bisphenol A impairs the reproductive axis of prepuberal male rats.

The objective of the present work was to study the effect of a low dose of bisphenol A (BPA), on the reproductive axis of prepuberal male rats exposed to the endocrine disruptor (ED) during gestation and lactation period. Wistar-mated rats were treated with either 0.1 % ethanol or BPA in their drinking water until their offspring were weaned at the age of 21 days. The estimated average dose of exposure to dams was approximately 3 µg/kg/day of BPA. The pups were sacrificed on the 35th day of life. Body weight was measured during the development and at the moment of the sacrifice; testicular and seminal vesicles weight and their respective relative weights were also measured. LH, FSH and testosterone were determined and histological studies of testicular tissue were also performed. Body weight at the moment of the sacrifice was significantly higher in the group exposed to BPA; testicular weight decreased significantly; seminal vesicles weight and relative weights of testes and seminal vesicles were not modified by treatment. LH and FSH serum levels increased significantly after treatment, meanwhile testosterone showed no significant changes. Histological studies showed the lumen of seminal tubes reduced by the presence of immature cells of the spermatic lineage. Our results suggest that pre- and early postnatal exposure to a low dose of BPA disrupts the normal function of the reproductive axis in prepuberal male rats. The effects of the ED may be exerted at different levels of the axis and may be dependent on the dose, manner of administration, and the moment of exposure to the disruptor.

Melatonin and diet-induced metabolic syndrome in rats: impact on the hypophysial-testicular axis.

Abstract Combinations of fructose- and fat-rich diets in experimental animals can model the human metabolic syndrome (MS). In rats, the increase in blood pressure (BP) after diet manipulation is sex related and highly dependent on testosterone secretion. However, the extent of the impact of diet on rodent hypophysial-testicular axis remains undefined. In the present study, rats drinking a 10% fructose solution or fed a high-fat (35%) diet for 10 weeks had higher plasma levels of luteinizing hormone (LH) and lower plasma levels of testosterone, without significant changes in circulating follicle-stimulating hormone or the weight of most reproductive organs. Diet manipulation brought about a significant increase in body weight, systolic BP, area under the curve (AUC) of glycemia after an intraperitoneal glucose tolerance test (IPGTT), and plasma low-density lipoprotein cholesterol, cholesterol, triglycerides, and uric acid levels. The concomitant administration of melatonin (25 µg/mL of drinking water) normalized the abnormally high LH levels but did not affect the inhibited testosterone secretion found in fructose- or high-fat-fed rats. Rather, melatonin per se inhibited testosterone secretion. Melatonin significantly blunted the body weight and systolic BP increase, the increase in the AUC of glycemia after an IPGTT, and the changes in circulating lipid profile and uric acid found in both MS models. The results are compatible with a primary inhibition of testicular function in diet-induced MS in rats and with the partial effectiveness of melatonin to counteract the metabolic but not the testicular sequelae of rodent MS.

Probable gamma-aminobutyric acid involvement in bisphenol A effect at the hypothalamic level in adult male rats.

The aim of the present study was to investigate the effects of bisphenol A (BPA) on the neuroendocrine mechanism of control of the reproductive axis in adult male rats exposed to it during pre- and early postnatal periods. Wistar mated rats were treated with either 0.1% ethanol or BPA in their drinking water until their offspring were weaned at the age of 21 days. The estimated average dose of exposure to dams was approximately 2.5 mg/kg body weight per day of BPA. After 21 days, the pups were separated from the mother and sacrificed on 70 day of life. Gn-RH and gamma-aminobutyric acid (GABA) release from hypothalamic fragments was measured. LH, FSH, and testosterone concentrations were determined, and histological and morphometrical studies of testis were performed. Gn-RH release decreased significantly, while GABA serum levels were markedly increased by treatment. LH serum levels showed no changes, and FSH and testosterone levels decreased significantly. Histological studies showed abnormalities in the tubular organization of the germinal epithelium. The cytoarchitecture of germinal cells was apparently normal, and a reduction of the nuclear area of Leydig cells but not their number was observed. Taken all together, these results provide evidence of the effect caused by BPA on the adult male reproductive axis when exposed during pre- and postnatal period. Moreover, our findings suggest a probable GABA involvement in its effect at the hypothalamic level.

Evidence to suggest glutamic acid involvement in Bisphenol A effect at the hypothalamic level in prepubertal male rats.

OBJECTIVES: The aim of present paper was to study the probable role of glutamic acid (GLU) as a mediator of bisphenol A (BPA) effect at the hypothalamic level and its effects on the reproductive axis of prepubertal male rats. METHODS: Mated Wistar rats were treated with either 0.1% ethanol (control group, n=10) or BPA (BPA group, n=10) in their drinking water until their offspring were weaned at the age of 21 days. The estimated average dose of exposure to dams was approximately 2.5 mg/kg body weight/day of BPA. At the prepubertal stage (35 days of age), the male rats were sacrificed and Gn-RH and glutamic acid (GLU) release, an amino acid involved in Gn-RH secretion, were measured in hypothalamic samples containing medio basal and anterior preoptic area (MBH-APOA), by RIA and HPLC respectively. LH, FSH serum levels were measured by RIA and testosterone by EQLIA. RESULTS: Gn-RH and GLU release decreased significantly in animals exposed to BPA (p<0.001, p<0.01). LH, FSH and testosterone serum levels were also decreased by treatment (p<0.0001). CONCLUSION: Present results provide evidence that BPA may act at the hypothalamic level to decrease GLU release which in turn may modify Gn-RH secretion altering the normal function of the axis.

Impact of gestational and lactational phthalate exposure on hypothalamic content of amino acid neurotransmitters and FSH secretion in peripubertal male rats.

This study investigated the effect of the pre- and perinatal exposure to di-(2-ethylhexyl) phthalate (DEHP) on the neuroendocrine parameters that regulate reproduction in peripubertal male rats. DEHP at dose of 3 and 30mg/kg bw/day was administered orally to female rat since pregnancy onset until weaning. The male litters were sacrificed at 30 days of age to determine gonadotropin serum level and the hypothalamic contents of the amino acids aspartate and gamma-aminobutyric acid. No changes in gonadotropin, aspartate and gamma-aminobutyric acid levels were detected at the low dose. DEHP 30mg/kg bw/day reduced testis weight and serum FSH, in correlation with a significant increase in the inhibitory GABAergic tone and a reduction in the stimulatory effect of aspartate on gonadotropin level. This study provides unknown data regarding changes in the hypothalamic contents of the amino acid neurotransmitters, which are involved in the neuroendocrine regulation of reproductive axis, in peripubertal male rat offspring from dams exposed to DEHP during gestational and lactational periods. This could be related with the gonadotropin modifications also here described.