ROTADIAL: The first nanobody-based immunoassay to detect Group A Rotavirus.

ROTADIAL is a rapid nanobody (Nb)-based ELISA assay able to identify Rotavirus group A (RVA) in feces from pediatric patients. The assay is based on a sandwich of two patented llama-derived Nbs directed to the inner capsid viral protein VP6 from RVA. Nbs are directed to conformational epitopes of VP6 and recognized all human RVA strains tested, representing ideal reagents for their use in immunodiagnostic tests for RVA detection. All the steps are carried out at room temperature, bringing results in less than two hours. This assay, named ROTADIAL, was validated with a reference panel of feces from pediatric patients from Argentina. The overall sensitivity and specificity of the ROTADIAL test, when compared to a commercial test, was 100 % (100/100) and 99 % (99/100) respectively. ROTADIAL presented optimal analytical performance, being capable of detecting RVA regardless of the presence of other common human enteric infectious agents and is the first RVA-diagnostic assay developed using Nbs, worldwide.

Influence of individual or group housing of newborn calves on rotavirus and coronavirus infection during the first 2 months of life.

Bovine rotavirus A (RVA) and bovine coronavirus (CoV) are the two main viral enteropathogens associated with neonatal calf diarrhea. The aim of the present work was to study the impact of group and individual housing systems in the epidemiology of RVA and CoV infection. Eleven calves reared in individual housing (FA) and nine calves in group housing (FB) were monitored during the first 7 weeks of life. Stool and serum samples were screened for RVA and CoV antigens by ELISA. IgG1 antibodies (Ab) to both antigens were also measured. From the 160 fecal samples collected, the proportion of positive samples to RVA and CoV was significantly higher in FB (23.6%) than in FA (9%) (p = 0.03). The geometric mean of colostral IgG1 Ab titers to CoV and RVA in FA (IgG1 anti-CoV 1024 and anti-RVA 1782.9) was lower than in FB (IgG1 anti-CoV 10,321.2 and anti-RVA 4096) at birth. Calves less than 2 weeks of life from FB had a higher risk of being infected by RVA (OR = 4.9; p = 0.01) and CoV (OR = 17.15; p = 0.01) than calves from FA. The obtained results showed that there was higher RVA and CoV shedding in group-housed calves than in individual-housed animals.

First report of group A rotavirus and bovine coronavirus associated with neonatal calf diarrhea in the northwest of Argentina.

Group A rotavirus (RVA) and bovine coronavirus (BCoV) are the two main viral enteropathogens associated with neonatal calf diarrhea. The aim of the present survey was to investigate the epidemiology and the role of RVA and BCoV in the presentation of dairy and beef calf diarrhea in Lerma Valley of Salta province, within the Northwest region of Argentina. Stool samples of calves with or without diarrhea younger than 2 months of age were collected from 19 dairy farms and 20 beef farms between the years 2014 and 2016. Stool samples were screened for RVA and BCoV detection by ELISA. Heminested multiplex RT-PCR was used for RVA typing and RT-PCR to confirm BCoV. Positive samples were submitted to sequencing analysis. Bovine RVA and BCoV were circulating in 63% (12/19) and 10.52% (2/19) of the dairy farms, respectively, where 9.5% (46/484) of the calves were positives to RVA and 0.4% (2/484) to BCoV. In beef herds, RVA was detected in 40% (8/20) of the farms and in 6.75% (21/311) of the calves, without positives cases of BCoV. Molecular analysis showed that in dairy farms, G6P[11] and G10P[11] were the prevalent RVA strains, while in beef farms, G10P[11] was the prevalent. The main finding was the detection for the first time of a G15P[11] causing diarrhea in beef calves of Argentina that represents a new alert to be consider for future vaccine updates. Analysis of detected BCoV showed that it is related to the other circulating strains of Argentina.

Prevalence and viability of group A rotavirus in dairy farm water sources.

AIM: To analyse group A rotavirus (RVA) environmental contamination in waters used for calves' consumption and to assess viral viability in dairy farm water sources. METHODS AND RESULTS: We analysed 202 samples of water used for calves' consumption and RVA was detected by RT-qPCR in 35·1% (95% CI: 28·9-42·0%). A marked pattern of seasonality was observed with higher frequency of detection in colder than warmer months (P = 0·002). There was no association between viral load and season or between the number of milking cows in the herd and the detection of RVA in the farm. The viability of the RVA particles detected was confirmed by isolation of RVA in cell culture from 5 of 10 water samples. Furthermore, an RVA waterborne outbreak of neonatal calf diarrhoea was described. CONCLUSIONS: We demonstrate that RVA is frequent in dairy farm waters, and that the virus is infectious and capable of generating a diarrhoea outbreak. SIGNIFICANCE AND IMPACT OF THE STUDY: Neonatal diarrhoea syndrome leads to economic losses to the livestock industry worldwide. To determine transmission routes is essential to take action in this regard and reduce the impact that this syndrome has for the livestock production. The results obtained in this work alert the dairy industry and highlight that mitigation strategies are crucial to improve the microbiological quality of this water.

An evaluation of cumulative risks from offshore produced water discharges in the Bass Strait.

Chemical analyses and toxicity testing using six marine species were used to characterize the hazard of produced waters (PW) to marine life from twelve Australian offshore platforms. Hazard data were used in conjunction with platform-specific plume discharge dilution and species sensitivity distribution modeling to estimate cumulative risks by calculating the multiple substance potentially affected fraction of species in the local marine environment. Results provided two independent lines of evidence demonstrating that cumulative risks to marine life from these discharges meet intended 95% species protection goals at the edge of the mixing zone. A limited number of PW constituents (hydrocarbons, sulphide and ammonia) appeared to dictate risk thereby informing management and providing a rationale for more targeted analyses in future monitoring studies. Based on these findings a tiered framework is proposed to foster consistent screening and potential refinement of cumulative risk evaluations for PW discharges.

Egg yolk IgY antibodies: A therapeutic intervention against group A rotavirus in calves.

Bovine group A rotavirus (RVA) is considered the major cause of diarrhea in intensively reared neonatal calves. Chicken egg yolk antibodies (IgY) are efficient in protecting neonatal calves from RVA diarrhea; however, the value of this intervention in calves once diarrhea has appeared is unclear. The aim of the present study was to evaluate the application of RVA-specific IgY as a passive treatment in those cases. The experimental groups were: G1=RVA-specific IgY treatment; G2=no Ab treatment; and G3=colostrum deprived+no Ab treatment. IgY treatment significantly reduced virus shedding, diarrhea duration and severity compared to G2 and G3 calves. However, it caused a partial suppression of systemic Ab responses to RVA that could be associated with less severe diarrhea. The oral treatment with IgY for 7days was associated with significantly higher antibody secreting cell responses in the calves compared with other groups of animals.

Molecular and antigenic characterization of bovine Coronavirus circulating in Argentinean cattle during 1994-2010.

Bovine coronavirus (BCoV) is an important viral pathogen associated with neonatal calf diarrhea. Our aim was to investigate the incidence of BCoV in diarrhea outbreaks in beef and dairy herds from Argentina during 1994-2010. A total of 5.365 fecal samples from diarrheic calves were screened for BCoV diagnosis by ELISA. The virus was detected in 1.71% (92/5365) of the samples corresponding to 5.95% (63/1058) of the diarrhea cases in 239 beef and 324 dairy farms. The detection rate of BCoV was significantly higher in dairy than in beef herds: 12.13% (29/239) vs. 4.32% (14/324) respectively. Phylogenetic analysis of the hypervariable S1 region of seven representative samples (from different husbandry systems, farm locations and years of sampling) indicated that BCoV strains circulating in Argentinean beef and dairy herds formed a cluster distinct from other geographical regions. Interestingly, Argentinean strains are distantly related (at both the nucleotide and amino acid levels) with the Mebus historic reference BCoV strain included in the vaccines currently available in Argentina. However, Mebus-induced antibodies were capable of neutralizing the BCoV Arg95, a field strain adapted to grow in vitro, and vice versa, indicating that both strains belong to the same CoV serotype reported in cattle. This work represents the first large survey describing BCoV circulation in Argentinean cattle.

Egg yolk IgY: protection against rotavirus induced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves.

Bovine rotavirus (BRV) is an important cause of diarrhea in newborn calves. Local passive immunity is the most efficient protective strategy to control the disease. IgY technology (the use of chicken egg yolk immunoglobulins) is an economic and practical alternative to prevent BRV diarrhea in dairy calves. The aim of this study was to evaluate the protection and immunomodulation induced by the oral administration of egg yolk enriched in BRV specific IgY to experimentally BRV infected calves. All calves in groups Gp 1, 2 and 3 received control colostrum (CC; BRV virus neutralization Ab titer - VN=65,536; ELISA BRV IgG(1)=16,384) prior to gut closure. After gut closure, calves received milk supplemented with 6% BRV-immune egg yolk [(Gp 1) VN=2048; ELISA IgY Ab titer=4096] or non-immune control egg yolk [(Gp 2) VN<4; ELISA IgY Ab titer<4] twice a day, for 14 days. Calves receiving CC only or colostrum deprived calves (CD) fed antibody (Ab) free milk served as controls (Gp 3 and 4, respectively). Calves were inoculated with 10(5.85)focus forming units (FFU) of virulent BRV IND at 2 days of age. Control calves (Gp 3 and 4) and calves fed control IgY (Gp 2) were infected and developed severe diarrhea. Around 80% calves in Gp 1 (IgY 4096) were infected, but they showed 80% (4/5) protection against BRV diarrhea. Bovine RV-specific IgY Ab were detected in the feces of calves in Gp 1, indicating that avian antibodies (Abs) remained intact after passage through the gastrointestinal tract. At post infection day 21, the duodenum was the major site of BRV specific antibody secreting cells (ASC) in all experimental groups. Mucosal ASC responses of all isotypes were significantly higher in the IgY treated groups, independently of the specificity of the treatment, indicating that egg yolk components modulated the immune response against BRV infection at the mucosal level. These results indicate that supplementing newborn calves' diets for the first 14 days of life with egg yolk enriched in BRV-specific IgY represents a promising strategy to prevent BRV diarrhea. Moreover a strong active ASC immune response is induced in the intestinal mucosa following BRV infection after the administration of egg yolk, regardless the specificity of the treatment.

The molecular genetics and evolution of colour and polarization vision in stomatopod crustaceans.

Stomatopod crustaceans have the most complex assemblage of visual receptor classes known; retinas of many species are thought to express up to 16 different visual pigments. Physiological studies indicate that stomatopods contain up to six distinct middle-wavelength-sensitive (MWS) photoreceptor classes, suggesting that no more than six different MWS opsin gene copies exist per species. However, we previously reported the unexpected expression of 6-15 different MWS genes in retinas of each of five stomatopod species (Visual Neurosci 26: 255-266, 2009). Here, we present a review of the results reported in this publication, plus new results that shed light on the origins of the diverse colour and polarization visual capabilities of stomatopod crustaceans. Using in situ hybridization of opsins in photoreceptor cells, we obtained new results that support the hypothesis of an ancient functional division separating spatial and polarizational vision from colour vision in the stomatopods. Since evolutionary trace analysis indicates that stomatopod MWS opsins have diverged both with respect to spectral tuning and to cytoplasmic interactions, we have now further analyzed these data in an attempt to uncover the origins, diversity and potential specializations among clades for specific visual functions. The presence of many clusters of highly similar transcripts suggests exuberant opsin gene duplication has occurred in the stomatopods, together with more conservative, ancient gene duplication events within the stem crustacean lineage. Phylogenetic analysis of opsin relatedness suggests that opsins specialized for colour vision have diverged from those devoted to polarization vision, and possibly motion and spatial vision.

Distribution of phosphate-activated glutaminase isozymes in the chicken: absence from liver but presence of high activity in pectoralis muscle.

The distribution of glutaminase expression in a uricotelic species, the chicken, has been examined using cDNA probes to the rat isozymes. The results suggest that chickens do not possess a glutaminase isozyme equivalent to the liver-type isozyme of mammalian liver. Measurements of enzymic activity also showed very low glutaminase activity in chicken liver. Extra-hepatic tissues in the chicken do express a glutaminase isozyme mRNA which is detected by rat kidney-type glutaminase cDNA. The abundance of this mRNA was highest in kidney and breast muscle and relatively abundant in brain, spleen and adipose tissue. Chicken small intestine expressed relatively low levels of the mRNA. The high level of glutaminase mRNA in chicken pectoralis muscle was accompanied by high glutaminase enzymic activity. In contrast, in mixed leg muscle glutaminase mRNA was barely detectable by Northern blot and glutaminase activity was relatively low. Starvation for 48 h resulted in a slight decrease in the activity of glutaminase in pectoralis muscle, but a large decrease in the relative abundance of the mRNA. The results suggest that in the chicken, hepatic glutamine hydrolysis is not quantitatively important, but skeletal muscle may be a major site of glutamine catabolism.

Rat hepatic glutaminase: identification of the full coding sequence and characterization of a functional promoter.

Glutamine catabolism in mammalian liver is catalysed by a unique isoenzyme of phosphate-activated glutaminase. The full coding and 5' untranslated sequence for rat hepatic glutaminase was isolated by screening lambda ZAP cDNA libraries and a Charon 4a rat genomic library. The sequence produces a mRNA 2225 nt in length, encoding a polypeptide of 535 amino acid residues with a calculated molecular mass of 59.2 kDa. The deduced amino acid sequence of rat liver glutaminase shows 86% similarity to that of rat kidney glutaminase and 65% similarity to a putative glutaminase from Caenorhabditis elegans. A genomic clone to rat liver glutaminase was isolated that contains 3.5 kb of the gene and 7.5 kb of the 5' flanking region. The 1 kb immediately upstream of the hepatic glutaminase gene (from -1022 to +48) showed functional promoter activity in HepG2 hepatoma cells. This promoter region did not respond to treatment with cAMP, but was highly responsive (10-fold stimulation) to the synthetic glucocorticoid dexamethasone. Subsequent 5' deletion analysis indicated that the promoter region between -103 and +48 was sufficient for basal promoter activity. This region does not contain an identifiable TATA element, indicating that transcription of the glutaminase gene is driven by a TATA-less promoter. The region responsive to glucocorticoids was mapped to -252 to -103 relative to the transcription start site.