Serum metabolome differences associated with subclinical intramammary infection caused by Streptococcus agalactiae and Prototheca spp. in multiparous dairy cows.

Mastitis is one of the most significant diseases in dairy cows and causes several economic losses. Somatic cell count (SCC) is often used as an indirect diagnostic tool for mastitis, especially for subclinical mastitis (SCM) where no symptoms or signs can be detected. Streptococcus agalactiae is one of the main causes of contagious mastitis, and Prototheca spp. is an alga-inducing environmental mastitis that is not always correlated with increased milk SCC. The aim of this study was to evaluate the changes in the metabolomic profile of blood in relation to subclinical intramammary infection (IMI) in dairy cows. In addition, differences resulting from the etiologic agent causing mastitis were also considered. Forty Holstein-Friesian dairy cows in mid and late lactation were enrolled in this cross-sectional design study. Based on the bacteriological examination of milk, the animals were divided into 3 groups: group CTR (control group; n = 16), group A (affected by SCM with IMI caused by Strep. agalactiae; n = 17), and group P (affected by SCM with IMI caused by Prototheca spp.; n = 7). Blood samples from the jugular vein were collected in tubes containing clot activator; the serum aliquot was stored until metabolomic analysis by 1H-nuclear magnetic resonance spectroscopy. Statistical analysis was conducted by fitting a linear model with the group as the fixed effect and SCC as the covariate. Forty-two metabolites were identified, and among them 10 were significantly different among groups. Groups A and P showed greater levels of His and lactose and lower levels of acetate, Asn, and dimethylamine compared with group CTR. Group A showed high levels of Val, and group P showed high levels of Cit and methylguanidine, as well as lower levels of 3-hydroxybutyrate, acetone, allantoin, carnitine, citrate, and ethanol. These metabolites were related to ruminal fermentations, energy metabolism, urea synthesis and metabolism, immune and inflammatory response, and mammary gland permeability. These results suggest systemic involvement with subclinical IMI and that the metabolic profile of animals with SCM undergoes changes related to the etiological agent of mastitis.

Quarter-level analyses of the associations among subclinical intramammary infection and milk quality, udder health, and cheesemaking traits in Holstein cows.

In this study, we investigated associations among subclinical intra-mammary infection (IMI) and quarter-level milk composition, udder health indicators, and cheesemaking traits. The dataset included records from 450 Holstein cows belonging to three dairy herds. After an initial screening (T0) to identify animals infected by Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, and Prototheca spp., 613 quarter milk samples for 2 different sampling times (T1 and T2, 1 mo after T1) were used for analysis. Milk traits were analyzed using a hierarchical linear mixed model including the effects of days in milk, parity and herd, and bacteriological and inflammatory category [culture negative with somatic cell count (SCC) <200,000 cells/mL; culture negative with SCC ≥200,000 cells/mL; or culture positive]. All udder health indicators were associated with increased SCC and IMI at both sampling times. The largest effects were detected at T2 for milk lactose (-7% and -5%) and milk conductivity (+9% and +8%). In contrast, the increase in differential SCC (DSCC) in samples with elevated SCC was larger at T1 (+17%). Culture-negative samples with SCC ≥200,000 cells/mL had the highest SCC and greatest numbers of polymorphonuclear-neutrophils-lymphocytes and macrophages at both T1 and T2. Regarding milk cheesemaking ability, samples with elevated SCC showed the worst pattern of curd firmness at T1 and T2. At T2, increased SCC and IMI induced large decreases in recoveries of nutrients into the curd, in particular recovered protein (-14% and -16%) and recovered fat (-12% and -14%). Different behaviors were observed between Strep. agalactiae and Prototheca spp., especially at T2. In particular, samples that were positive for Strep. agalactiae had higher proportions of DSCC (+19%) compared with negative samples with low SCC, whereas samples that were positive for Prototheca spp. had lower DSCC (-11%). Intramammary infection with Prototheca spp. increased milk pH compared with culture-negative samples (+3%) and negative samples that had increased SCC (+2%). The greatest impairment in curd firmness at 30 min from rennet addition was observed for samples that were positive for Prototheca spp. (-99% compared with negative samples, and -98% compared with negative samples with high SCC). These results suggest that IMI caused by Prototheca spp. have detrimental effects on milk technological traits that deserve further investigation of the mechanisms underlying animals' responses to infection.

Active and passive surveillance for bat lyssaviruses in Italy revealed serological evidence for their circulation in three bat species.

The wide geographical distribution and genetic diversity of bat-associated lyssaviruses (LYSVs) across Europe suggest that similar viruses may also be harboured in Italian insectivorous bats. Indeed, bats were first included within the passive national surveillance programme for rabies in wildlife in the 1980s, while active surveillance has been performed since 2008. The active surveillance strategies implemented allowed us to detect neutralizing antibodies directed towards European bat 1 lyssavirus in six out of the nine maternity colonies object of the study across the whole country. Seropositive bats were Myotis myotis, M. blythii and Tadarida teniotis. On the contrary, the virus was neither detected through passive nor active surveillance, suggesting that fatal neurological infection is rare also in seropositive colonies. Although the number of tested samples has steadily increased in recent years, submission turned out to be rather sporadic and did not include carcasses from bat species that account for the majority of LYSVs cases in Europe, such as Eptesicus serotinus, M. daubentonii, M. dasycneme and M. nattereri. A closer collaboration with bat handlers is therefore mandatory to improve passive surveillance and decrypt the significance of serological data obtained up to now.

Regulation of neuronal plasticity and fear by a dynamic change in PAR1-G protein coupling in the amygdala.

Fear memories are acquired through neuronal plasticity, an orchestrated sequence of events regulated at circuit and cellular levels. The conventional model of fear acquisition assumes unimodal (for example, excitatory or inhibitory) roles of modulatory receptors in controlling neuronal activity and learning. Contrary to this view, we show that protease-activated receptor-1 (PAR1) promotes contrasting neuronal responses depending on the emotional status of an animal by a dynamic shift between distinct G protein-coupling partners. In the basolateral amygdala of fear-naive mice PAR1 couples to Gαq/11 and Gαo proteins, while after fear conditioning coupling to Gαo increases. Concurrently, stimulation of PAR1 before conditioning enhanced, but afterwards it inhibited firing of basal amygdala neurons. An initial impairment of the long-term potentiation (LTP) in PAR1-deficient mice was transformed into an increase in LTP and enhancement of fear after conditioning. These effects correlated with more frequent 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) receptor-mediated miniature post synaptic events and increased neuronal excitability. Our findings point to experience-specific shifts in PAR1-G protein coupling in the amygdala as a novel mechanism regulating neuronal excitability and fear.

Effects of the provision of large amounts of solid feeds to veal calves on growth and slaughter performance and intravitam and postmortem welfare indicators.

Polish Friesian male calves (n = 78) were used to investigate the effects of innovative feeding plans based on the provision of large amounts of solid feeds on growth performance, welfare, and carcass characteristics of veal calves. Groups of calves (initial BW 71.5 ± 3.7 kg) were fed 1 of 3 treatments (26 calves/treatment): 1) milk replacer plus corn grain (CG), 2) milk replacer plus an 80:20 mixture (as-fed basis) of corn grain and wheat straw (CGS), or 3) milk replacer plus a 72:20:8 mixture (as-fed basis) of corn grain, wheat straw, and extruded soybean, respectively (CGSES). All the treatments provided at least 170 kg DM/calf from solid feed throughout the 206 d of fattening. Type and concentration of milk replacer were the same for all calves throughout fattening. Calves fed CGSES received 96% of the daily amount of milk replacer delivered to CG and CGS to balance the dietary CP content. No differences (P ≥ 0.063) among treatments were observed for growth performance and DMI. Health status of CG calves was less than CGSES calves, as indicated by the greatest (P < 0.001) proportion of milk replacer refusal events and the greatest (P < 0.001) incidence of treatments for respiratory disorders. Inclusion of straw and soybean in the solid feeds increased (P < 0.001) the daily intake of iron in CGS and CGSES as compared with CG; however, blood hemoglobin concentrations measured at d 5 and 31 were greater (P < 0.05) than when measured on d 80, 122, and 206 of fattening. Feeding treatment did not (P ≥ 0.107) affect HCW, dressing percentage, or carcass color. Calves fed CG had heavier (P < 0.001) reticulorumens and more (P < 0.001) developed rumens than CGS and CGSES calves, but 84% and 68% of CG rumens (P < 0.001) showed overt signs of hyperkeratinization and plaques, respectively. These alterations of rumen mucosa were not detected in CGSES calves, and only 8% of CGS calves had rumen plaques. Regardless of feeding treatment, postmortem inspection recorded a prevalence of more than 84% abomasal lesions. When feeding veal calves with large amounts of solids, it is advisable to avoid the provision of corn grain alone and replace part of the cereal with a roughage source to improve calf health and prevent rumen mucosa alterations.

Voltage-dependent Na(v)1.7 sodium channels: multiple roles in adrenal chromaffin cells and peripheral nervous system.

Voltage-dependent Na+ channels consist of the principal alpha-subunit (approximately 260 kDa), without or with auxiliary beta-subunit (approximately 38 kDa). Nine alpha-subunit isoforms (Na(v)1.1-Na(v)1.9) are encoded in nine different genes (SCN1A-SCN5A and SCN8A-SCN11A). Besides initiating and propagating action potentials in established neuronal circuit, Na+ channels engrave, maintain and repair neuronal network in the brain throughout the life. Adrenal chromaffin cells express Na(v)1.7 encoded in SCN9A, which is widely distributed among peripheral autonomic and sensory ganglia, neuroendocrine cells, as well as prostate cancer cell lines. In chromaffin cells, Na(v)1.7-specific biophysical properties have been characterized; physiological stimulation by acetylcholine produces muscarinic receptor-mediated hyperpolarization followed by nicotinic receptor-mediated depolarization. In human patients with Na(v)1.7 channelopathies, gain-of-pathological function mutants (i.e. erythermalgia and paroxysmal extreme pain disorder) or loss-of-physiological function mutant (channelopathy-associated insensitivity to pain) proved the causal involvement of mutant Na(v)1.7 in generating intolerable pain syndrome, Na(v)1.7 being the first molecular target convincingly identified for pain treatment. Importantly, aberrant upregulation/hyperactivity of even the native Na(v)1.7 produces pain associated with inflammation, nerve injury and diabetic neuropathy in rodents. Various extra- and intracellular signals, as well as therapeutic drugs modulate the activity of Na(v)1.7, and also cause up- and downregulation of Na(v)1.7. Na(v)1.7 seems to play an increasing number of crucial roles in health, disease and therapeutics.

Produção deficiente de citocinas Th1 em camundongos BALB/c jovens / Deficient production of Th1 cytokines in young BALB/c mice

O objetivo do presente estudo foi comparar a produção de IFN-gama , IL-12 e IL-4 entre camundongos jovens (5, 12e 19 dias de idade) e adultos (30 dias de idade). As avaliações foram feitas por estimulação, in vitro, de células esplênicas com Concanavalina A (ConA) , Staphylococcus aureus (S. aureus) e lipopolissacarídeo (LPS). Diferentes concentrações de cada estímulo foram testadas e os sobrenadantes das culturas foram coletados após 48 horas de incubação e as concentrações de IFN-gama IL-12 e IL-4 determinadas por ELISA. Células de camundongos jovens e adultos produziram níveis igualmente elevados de IFN-gama após estímulo com ConA. Somente animais adultos produziram IFN-gama em resposta ao estímulo com S. aureus. Em culturas estimuladas com LPS, a produção desta citocina foi baixa e similar nos animais jovens e significativamente elevada nos animais adultos. Somente células de animais adultos estimuladas com S. aureus foram capazes de produzir IL-12. O único estímulo capaz de induzir níveis detectáveis de IL-4 foi ConA, sendo que estes níveis foram mais elevados nos animais com 12 e 19 dias de idade em comparação com animais neonatos e adultos. A diminuição das doses ótimas dos estímulos não mudou o perfil de produção de cada citocina nos animais jovens. Estes resultados permitem concluir que a idade afeta a produção de citocinas: ocorre maior produção de IL-4 em camundongos jovens e maior produção de IL-12 e IFN-gama em animais adultos. Estas informações são importantes devido ao papel destas citocinas na polarização das respostas imunes nos sentidos Th1 e Th2.

Hepatitis B core antigen in serum during acute hepatitis B.

Hepatitis B core antigen was measured in sera of patients with acute and chronic hepatitis B virus infection by a modified radioimmunoassay based on high molarity treatment of samples to avoid masking of antigen by homologous antibody. A good correlation between hepatitis B core antigen levels and serum HBV-DNA was observed in sera obtained during chronic infection. In contrast, acute phase sera were often HBcAg positive but HBV-DNA negative, particularly when obtained during maximum liver damage. Sequential studies in 5 patients with acute hepatitis B showed that HBcAg positivity persisted beside HBV-DNA clearance and was often enhanced at the time of maximum liver damage, suggesting release of antigen from infected hepatocytes undergoing immunolysis, even after termination of virus replication.

Cell-mediated immunity to HBcAg in chronic HBV infection.

The role of hepatitis B core antigen (HBcAg) as a possible target of cell-mediated immune response in chronic hepatitis B virus (HBV) infection has been recently emphasized. Peripheral blood leukocytes (PBLs) from 35 chronic carriers of hepatitis B surface antigen (HBsAg) were studied in vitro for their immune response to a purified preparation of HBcAg isolated from circulating Dane particles. PBLs from all the studied HBsAg-positive patients yielded a stimulation index above 3, with values ranging from 3.1 to 38.1. None of the healthy seronegative subjects, taken as control group, had a stimulation index above 2, with a mean value +/- SD of 1.28 +/- 0.35. Levels of PBL stimulation correlated with the histologic activity of liver disease, and the differences reached statistical significance. These results indicate that lymphocyte response to HBcAg may be relevant in determining liver cell damage.

Detection of hepatitis B virus DNA sequences in bone marrow of children with leukemia.

To investigate the possibility that hemopoietic cells may become infected with hepatitis B virus (HBV), viral DNA was studied by molecular hybridization in bone marrow aspirates of 51 children with leukemia. HBV-DNA was found in the bone marrow of eight children (15%) and Southern blot analysis revealed the presence of free, monomeric viral sequences. Only one of the eight children with HBV-DNA in bone marrow cells was HBsAg-positive in serum, whereas two additional patients were transiently HBsAg-positive in serum during follow-up, but were negative at the time HBV-DNA was found in bone marrow. Four other cases developed antibodies to HBV. Cases of myeloid leukemia were more frequently positive for HBV-DNA in bone marrow (55%), compared with cases of lymphoid leukemia (7%). These results indicate that hemopoietic cells are susceptible to infection with hepatitis B virus and stimulate new interest into the relation of HBV infection to the development of some forms of leukemia, as four of eight cases of myeloid leukemia were HBV-DNA positive in bone marrow aspirates at diagnosis, prior to receiving any transfusion therapy.

Changes in serum hepatitis B virus (HBV) DNA positivity in chronic HBV infection: results of a long-term follow-up study of 138 patients.

In an assessment of the clinical relevance of serum hepatitis B virus (HBV) DNA testing in chronic HBV infection, changes in the presence of this marker were investigated by spot hybridization in 138 hepatitis B surface antigen (HBsAg)-positive patients with chronic liver disease who were followed up for one to eight years. Forty-one patients were treated with steroids, often with evidence of potentiation of viral replication, whereas 92 patients remained untreated and had no evidence of sigma agent infection during follow-up. Data analysis in these patients allowed us to determine the significance of testing for hepatitis B e antigen and for HBV DNA in the natural history of the infection. The findings indicate that sequential testing for serum HBV DNA may be of great importance in HBsAg chronic carriers with liver disease for adequate evaluation of HBV replication and for the contribution of HBV DNA to the clinical assessment of chronic hepatitis.

Monoclonal radioimmunoassay for hepatitis B surface antigen in sera of children with acute leukemia.

Hepatitis B surface antigen (HBsAg) was detected by a monoclonal antibody radioimmunoassay in sera from five of 43 children (11.6%) with acute leukemia, who were negative by conventional assay. None of the nine positive sera had evidence of reactivity for HBV-DNA or DNA-polymerase activity. No correlation was found between the presence of HBsAg in serum by monoclonal RIA and the behaviour of anti-viral antibodies. Twenty-two children could be studied for liver HBsAg by immunofluorescence, and nine of them (40.9%) were positive, including three patients having HBsAg reactivity in serum. These data indicate that monoclonal antibodies increase the sensitivity of RIA for the detection of serum HBsAg in children with acute leukemia, who previously have frequently been found to have an atypical hepatitis B virus (HBV) serology.

Natural killer activity in patients with acute viral hepatitis.

Using the natural killer (NK) sensitive K562 cell line, enhanced NK cell cytotoxicity was demonstrable early in the course of acute hepatitis B while normal values were obtained in patients studied during convalescence. No evidence of enhanced NK activity was instead obtained in the course of acute non-A, non-B hepatitis. Serum levels of alpha-interferon, as determined by radioimmunoassay (RIA), were significantly increased in patients with acute hepatitis B showing enhanced NK cell activity but not in those with acute non-A, non-B hepatitis and normal NK cell activity. These results suggest that natural cytotoxicity may play a role early in the course of acute hepatitis type B, before antigen-specific T lymphocytes become fully operative.

Serum hepatitis B virus DNA in acute hepatitis type B.

Sera obtained within 7 days after clinical onset of acute hepatitis type B were positive for hepatitis B virus (HBV) DNA by spot hybridization only in 4 out of 45 patients who subsequently recovered, but in 10 out of 10 patients who instead developed chronic infection. These results indicate that in uncomplicated acute hepatitis B, virus replication is limited to an early phase of infection, often preceding the onset of clinical symptoms, and suggest that serum HBV-DNA may represent an early and predictive marker of chronicity.

Antibody to the hepatitis B virus receptor for polymerized albumin in acute infection and in hepatitis B vaccine recipients.

A receptor for polymerized human serum albumin is encoded by the pre-S region of the hepatitis B virus genome and may mediate attachment of the virion to hepatocytes. To investigate antibody response to the virus receptor we studied sera and their IgG fractions for inhibitory activity on hemagglutination of polyalbumin-coated red cells by virus particles containing the pre-S polypeptide. By this method antibody to the receptor was detected in serum in a goat immunized with pre-S containing particles, with no relation to levels of antibody to hepatitis B surface antigen, and in the sera of 33% and 83%, respectively, of acute hepatitis B patients studied during the early phase of illness and during convalescence. In contrast, antibody to the receptor was not detected in serum in any of the 47 subjects immunized with a commercial, plasma-derived, hepatitis B vaccine. These results demonstrate that natural acute infection with hepatitis B virus leads to production of antibody to the virus receptor for polyalbumin, while such antibody response is absent after immunization with currently licensed hepatitis B vaccines.

Hepatitis B core antigen (HBcAg) in serum of patients with chronic hepatitis B detected by a modified radioimmunoassay.

Serum hepatitis B core antigen (HBcAg) was investigated in 85 patients with chronic hepatitis B virus (HBV) infection using a modified radioimmunoassay technique, based on high molarity treatment of samples to avoid masking of the antigen by the excess homologous antibody. Eighty-eight percent of HBeAg-positive cases and 19% of anti-HBe-positive cases were HBcAg positive in serum, with a positive correlation with the presence of HBcAg in the liver. Although the sensitivity of the method for the presence of complete virions was not absolute, as shown by the comparison with serum HBV-DNA testing, this technique may be helpful for assessing virus synthesis in patients with HBV infection.