Search

To genetically assess the Australian distribution and frequency of Eimeria species in wild rabbits, with a primary focus on Eimeria intestinalis and Eimeria flavescens as possible additional agents of rabbit biocontrol, the distal colon and faecal samples from wild rabbits sourced from 26 Australian locations with mean annual rainfalls of between 252 mm and 925 mm were analysed using amplicon sequencing of the ITS1 region. Contrary to previous microscopy studies which had only detected E. flavescens on mainland Australia at Wellstead in south-west Western Australia, we detected this species at all 23 effectively sampled sites. The more pathogenic E. intestinalis was only found at 52.2% of sites. Three unique Eimeria genotypes were detected that did not align to the 11 published sequences using a pairwise-match threshold of 90%, and may represent unsequenced known species or novel species. One genotype we termed E. Au19SH and was detected at 20 sites, E. Au19CO was detected at eight sites, and E. Au19CN was detected in one rabbit at Crows Nest (Qld). Site diversity ranged from only five Eimeria species at Boboyan (ACT) to 13 unique sequences at Cargo (NSW). Eimeria diversity in individual rabbits ranged from 11 unique sequences in a rabbit at Wellstead (WA) and a rabbit at Cargo (NSW), to one in 17 rabbits and zero in six rabbits. The three rabbit age classes averaged 4.3 Eimeria species per rabbit. No relationship was found between the number of Eimeria species detected and mean annual rainfall. As Eimeria species were found to be fairly ubiquitous at most sites they appear to be an unlikely additional candidate to assist the control of pest rabbits in Australia.

Background: The occurrence of trans-placental transmission of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infection remains highly debated. Placental positivity for SARS-CoV-2 has been reported in selected cases, but infection or virus-associated disease of fetal tissues or newborns remains to be demonstrated. Methods: We screened for SARS-CoV-2 spike (S) protein expression placentas from 101 women who delivered between February 7 and May 15 2020, including 15 tested positive for SARS-CoV-2 RNA, 34 tested negative, and 52 not evaluated as they did not meet testing criteria (32), or delivered before COVID-19 pandemic declaration (20). Immunostain for SARS-CoV-2 nucleocapside (N) was performed in the placentas of all COVID-19 positive women. The single placenta resulted positive for the SARS-Cov2 S and N proteins was further studied by RNA-in situ hybridization for S transcripts, RT-PCR RNA, and by electron microscopy. A detailed immunohistochemical and immunofluorescence analysis of the placental inflammatory infiltrate completed the investigations. Findings: SARS-CoV2 S and N proteins were strongly expressed in the placenta of a COVID-19 pregnant woman whose newborn tested positive for viral RNA and developed COVID-19 pneumonia soon after birth. SARS-CoV-2 antigens, RNA and/or viral particles were identified in villous syncytiotrophoblast, endothelial cells, fibroblasts, in maternal macrophages, and in Hofbauer cells and fetal intravascular mononuclear cells. The placenta intervillous inflammatory infiltrate was composed of neutrophils and monocyte-macrophages displaying activation markers. Absence of villitis was associated with an increase in the number of Hofbauer cells, which expressed PD-L1. Scattered neutrophil extracellular traps (NETs) were identified by immunofluorescence. Interpretation: We provide first-time evidence for maternal-fetal transmission of SARS-CoV-2, likely mediated by circulating virus-infected fetal mononuclear cells. Placenta infection was associated with recruitment of maternal inflammatory cells in the intervillous space, without villitis. PD-L1 expression in syncytiotrophoblast and Hofbaeur cells, together with limited production of NETs, may have prevented immune cell-driven placental damage, ensuring sufficient maternal-fetus exchanges. Funding Statement: None. Declaration of Interests: All authors declare no competing interests. Ethics Approval Statement: The study was reviewed and approved by the Institutional Ethical Board of Brescia Hospital (ASST Spedali Civili Brescia), protocol numbers: NP 4151- Study COVID-BIO (Multicentric observational prospective study on SARS-CoV-2 infection during pregnancy) and NP 4133- Study INTERCOVID (A prospective cohort study of the effects of COVID-19 in pregnancy and the neonatal period).

Porcine epidemic diarrhoea virus (PEDV), belongs to the genus Alphacoronavirus in the family Coronaviridae and causes severe diarrhoea, vomiting, dehydration and high mortality in seronegative newborn piglets. Thus, a precise and rapid diagnosis of PEDV infection is important for the application of control measures to limit viral dissemination. In this investigation, a monoclonal antibodies (MAbs)-based competitive enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against PEDV was developed and validated. The diagnostic performance of the test was evaluated by receiver operating characteristic (ROC) analysis using a panel of 829 known sera collected from different commercial pig farms, with or without a history of PED presence and from experimentally infected pigs. The competitive ELISA showed excellent diagnostic performance and discriminatory power with high sensitivity (Se) and specificity (Sp) values (Se = 96.5%, 95% IC 94.1–98.1; Sp = 98.2%, 95% IC 96.3–99.3). Moreover, this competitive ELISA method has other properties, such as high feasibility of testing sera without pre-treatment and automatic and instrument-mediated revealing, that makes it appropriate for large-scale screenings of affected pig farms in endemic regions or for monitoring plans in PEDV-free areas., Highlights • A monoclonal antibodies (MAbs)-based competitive ELISA for detecting antibodies against PEDV was developed and validated. • The diagnostic performance of the test was evaluated by ROC analysis using a panel of 829 known sera. • The ELISA showed excellent diagnostic performance and discriminatory power.

The vesivirus strain 2117 has been identified as contaminant of bioreactors used for production of human drugs, due to possible contamination of the reagents used for cell cultivation. Using an ELISA assay, antibodies specific for 2117-like viruses were detected in 32/410 (7.8%) human sera, indicating exposure to these viruses.

Usutu virus is an arbovirus closely related to West Nile virus (genus Flavivirus), which circulates between mosquitoes and wild birds. This virus has been increasingly reported in Europe, raising concerns for its possible pathogenic potential for wild birds and humans. This study reports the whole genome sequences of 15 strains of USUV, isolated between 2010 and 2014 from mosquitoes and wild birds in the course of West Nile virus surveillance in the Emilia-Romagna and Lombardy regions of Northern Italy. Both whole and 656 partial genome sequences, obtained from isolated viruses and field samples (mosquitoes and wild birds), were analyzed to describe the temporal and geographical spread of USUV in the surveyed area. The detected sequences belonged to two groups, with one circulating primarily in the northwestern part of the area and the other in the southeastern part. This pattern is likely the result of different routes of introduction from the North (over the Alps) and from the East, respectively. The phylogenetic analysis of obtained sequences and other European sequences demonstrated that the majority of European strains belonged to one main clade, while less common strains, mainly from Western Europe, fell in other two clades. This analysis strongly suggested an autochthonous evolution process of strains of the main clade from a common ancestor with an estimated time of arrival in Europe at the beginning of the 1990s. In addition to causing mass mortality in wild birds, Usutu virus can infect humans and can sporadically cause disease. These factors and the endemization of the Usutu virus in a large area of Europe, sustained by the obtained data, strongly support the need to adequately survey Usutu virus in areas in which its circulation is detected.

Background Several functional size measurement methods have been proposed. A few ones –like IFPUG and COSMIC methods– are widely used, while others –like Simple Function Points method– are interesting new proposals, which promise to deliver functional size measures via a faster and cheaper measurement process. Objectives Since all functional size measurement methods address the measurement of the same property of software (namely, the size of functional specifications), it is expected that measures provided in a given measurement unit can be converted into a different measurement unit. In this paper, convertibility of IFPUG Function Points, COSMIC Function Points, and Simple Function Points is studied. Method Convertibility is analyzed statistically via regression techniques. Seven datasets, each one containing measures of a set of software applications expressed in IFPUG Function Points, COSMIC Function Points and Simple Function Points, were analyzed. The components of functional size measures (usually known as Base Functional Components) were also involved in the analysis. Results All the analyzed measures appear well correlated to each other. Statistically significant quantitative models were found for all the combinations of measures, for all the analyzed datasets. Several models involving Base Functional Components were found as well. Conclusions From a practical point of view, the paper shows that converting measures from a given functional size unit into another one is viable. The magnitude of the conversion errors is reported, so that practitioners can evaluate if the expected conversion error is acceptable for their specific purposes. From a conceptual point of view, the paper shows that Base Functional Components of a given method can be used to estimate measures expressed in a different measurement unit: this seems to imply that different functional size measurement methods are ‘structurally’ strongly correlated.

Measuring Function Points following the standard process is sometimes long and expensive. To solve this problem, several early estimation methods have been proposed. Among these, the “NESMA Estimated” method is one of the most widely used; it has also been selected by the International Function Point User Group as the official early function point analysis method, under the name of ‘High-level FPA’ method. A large-scale empirical study has shown that the High-level FPA method – although sufficiently accurate – tends to underestimate the size of software. Underestimating the size of the software to be developed can easily lead to wrong decisions, which can even result in project failure. In this paper we investigate the reasons why the High-level FPA method tends to underestimate. We also explore how to improve the method to make it more accurate. Finally, we propose size estimation models built using different criteria and we evaluate the estimation accuracy of these new models. Our results show that it is possible to derive size estimation models from historical data using simple regression techniques: these models are slightly less accurate than those delivered by the High-level FPA method in terms of absolute estimation errors, but can be used earlier than the High-level FPA method, are cheaper, and do not underestimate software size.

Human cerebral organoids (HCOs) are an in vitro model of early neural development, aimed at modelling and understanding brain development and neurological disorders. In just a few years there has been rapid and considerable progress in the attempt to create a brain model capable of showcasing the characteristics of the human brain. There are still strong limitations to address, including the absence of vascularization which makes it difficult to feed the central layers of the organoid. Nevertheless, some important features of the nervous system have recently been observed in cerebral organoids: they manifest electrical activity (i.e. communication between neurons), are sensitive to light stimulation and are able to connect to a spinal cord by sending impulses that make a muscle contract. Recent data show that cortical organoid network development at ten months resembles some preterm babies EEG patterns. Although cerebral organoids are not close to human brains so far due to their extremely simplified structure, this state of things gives rise to ethical concerns about the creation and destructive experimental use of human cerebral organoids. Particularly, one can wonder whether a human cerebral organoid could develop some degree of consciousness and whether, under certain conditions, it could acquire its own moral status with the related rights. In this article, I discuss the conditions under which HCOs could be granted their own moral status. For this purpose, I consider the hypothesis that HCOs might develop a primitive form of consciousness and investigate the ways in which it could be detected. In light of all this, I finally point out some cautionary measures that could be introduced into research on and with human cerebral organoids.

In a recent work, our group showed the existence of two distinct mesenchymal stem cell (MSC) subsets within human umbilical cord blood. One less proliferative and short-living (SL-CBMSC), the other with higher growth rate and long-living (LL-CBMSC), and therefore better suited for regenerative medicine applications. We examined whether LL-CBMSC possess peculiar paracrine properties able to affect angiogenesis or inflammatory processes. It was shown for the first time that pro-angiogenic, proliferation-stimulating and tissue repairing factors were released at high level not only as soluble cytokines, but also as mRNA precursors embedded in membrane vesicles. The combination of this primary (proteic factors interacting with surface receptors) and delayed (mRNA transferred and translated via vesicle fusion and cargo release) interaction in endothelial target cells resulted in strong blood vessel induction with the development of capillary-like structures. In addition, LL-CBMSC dynamically modulated their release of pro-angiogenic and anti-inflammatory factors in an in vitro model of damage. In conclusion, LL-CBMSC synthesize and secrete multiple factors that may be attuned in response to the status of the target cell, a crucial requisite when paracrine mechanisms are needed at onset of tissue regeneration.

This study determined the seroprevalence of Encephalitozoon cuniculi in pet rabbits from north-central Italy and correlated it with data (age and clinical signs) obtained from private veterinary practitioners. During the period 2007 to 2010, 826 pet rabbit sera were collected and tested using carbon immunoassay. In total, 310 out of 826 rabbits (37.53%, 95% CI: 34.23 to 40.94) displayed clinical signs consistent with infection, whereas the remaining 516 animals presented for wellness examination or to be vaccinated. The results were analyzed according to age, serological status, and the presence or absence of clinical signs. Seropositive results were observed in 59.56% (95% CI: 56.12 to 62.92) of the examined animals. Seroprevalence in the clinically ill rabbits was 70.65% (95% CI: 65.18 to 75.59), much higher than in healthy animals, which showed a prevalence of 52.91% (95% CI: 48.5 to 57.27). Clinical signs were classified into 4 groups: nonvestibular neurologic signs, vestibular signs (including head tilt), renal failure, and ocular lesions. Odds of animals presenting with clinical disease signs were higher in seropositive rabbits (odds ratio=2.14, 95% CI: 1.59 to 2.9), and this association was particularly true for vestibular signs (odds ratio=4.1, 95% CI: 2.48 to 7.13). The prevalence values were different with regard to age, with a peak in 3- to 4-year-old animals. Average titers in both healthy and clinically ill rabbits at various ages were different; rabbits with overt disease signs had higher titers when very young ( E. cuniculi in pet rabbits in Italy, underlying growing interest and concern among veterinarians and pet owners regarding this organism.

Background Adult skin fibroblasts represent the most common starting cell type used to generate human induced pluripotent stem cells (F-hiPSC) for clinical studies. Yet, a foetal source would offer unique advantages, primarily the absence of accumulated somatic mutations. Herein, we generated hiPSC from cord blood multipotent mesenchymal stromal cells (MSC-hiPSC) and compared them with F-hiPSC. Assessment of the full activation of the pluripotency gene regulatory network (PGRN) focused on circular RNA (circRNA), recently proposed to participate in the control of pluripotency. Methods Reprogramming was achieved by a footprint-free strategy. Self-renewal and pluripotency of cord blood MSC-hiPSC were investigated in vitro and in vivo, compared to parental MSC, to embryonic stem cells and to F-hiPSC. High-throughput array-based approaches and bioinformatics analyses were applied to address the PGRN. Findings Cord blood MSC-hiPSC successfully acquired a complete pluripotent identity. Functional comparison with F-hiPSC showed no differences in terms of i) generation of mesenchymal-like derivatives, ii) their subsequent adipogenic, osteogenic and chondrogenic commitment, and iii) their hematopoietic support ability. At the transcriptional level, specific subsets of mRNA, miRNA and circRNA (n = 4,429) were evidenced, casting a further layer of complexity on the PGRN regulatory crosstalk. Interpretation A circRNA map of transcripts associated to naive and primed pluripotency is provided for hiPSC of clinical-grade foetal origin, offering insights on still unreported regulatory circuits of the PGRN to consider for the optimization and development of efficient differentiation protocols for clinical translation. Funding This research was funded by Ricerca Corrente 2012-2018 by the Italian Ministry of Health.

Bocaparvovirus is a newly established genus within the family Parvoviridae and has been identified as a possible cause of enteric, respiratory, reproductive/neonatal and neurological disease in humans and several animal species. In this study, metagenomic analysis was used to identify and characterise a novel bocaparvovirus in the faeces of rabbits with enteric disease. To assess the prevalence of the novel virus, rectal swabs and faecal samples obtained from rabbits with and without diarrhoea were screened with a specific PCR assay. The complete genome sequence of the novel parvovirus was reconstructed. The virus was distantly related to other bocaparvoviruses; the three ORFs shared 53%, 53% and 50% nucleotide identity, respectively, to homologous genes of porcine bocaparvoviruses. The virus was detected in 8/29 (28%) and 16/95 (17%) samples of rabbits with and without diarrhoea, respectively. Sequencing of the capsid protein fragment targeted by the diagnostic PCR identified two distinct bocaparvovirus populations/sub-types, with 91.7-94.5% nucleotide identity to each other. Including these novel parvoviruses in diagnostic algorithms of rabbit diseases might help inform their potential pathogenic role and impact on rabbit production and the virological profiles of laboratory rabbits.

Context: Functional size measurement methods are widely used but have two major shortcomings: they require a complete and detailed knowledge of user requirements, and they involve relatively expensive and lengthy processes. Objective: UML is routinely used in the software industry to effectively describe software requirements in an incremental way, so UML models grow in detail and completeness through the requirements analysis phase. Here, we aim at defining the characteristics of increasingly more refined UML requirements models that support increasingly more sophisticated - hence presumably more accurate - size estimation processes. Method: We consider the COSMIC method and three alternative processes (two of which are proposed in this paper) to estimate COSMIC size measures that can be applied to UML diagrams at progressive stages of the requirements definition phase. Then, we check the accuracy of the estimates by comparing the results obtained on a set of projects to the functional size values obtained with the standard COSMIC method. Results: Our analysis shows that it is possible to write increasingly more detailed and complete UML models of user requirements that provide the data required by COSMIC size estimation methods, which in turn yield increasingly more accurate size measure estimates of the modeled software. Initial estimates are based on simple models and are obtained quickly and with little effort. The estimates increase their accuracy as models grow in completeness and detail, i.e., as the requirements definition phase progresses. Conclusion: Developers that use UML for requirements modeling can obtain an early estimation of the application size at the beginning of the development process, when only a very simple UML model has been built for the application, and can obtain increasingly more accurate size estimates while the knowledge of the product increases and UML models are refined accordingly.