Investigating on Besnoitia besnoiti (Apicomplexa, Sarcocystidae) in naturally infected dairy cattle by an integrated approach.

Bovine besnoitiosis, caused by Besnoitia besnoiti, is a (re)emerging disease in Europe (Cortes et al., 2014), including Italy (Gazzonis et al., 2014; 2017). However, its economic impact is scarcely considered and generally underestimated and there are still little studied aspects concerning both the parasite and the disease. Following a natural outbreak of besnoitiosis in a dairy herd, a study was planned to characterize B. besnoiti infection in cattle through a multidisciplinary approach.Suspicious abortions and clinical cases of besnoitiosis were reported in a dairy farm (September 2017, Northern Italy) housing 216 Holstein cattle. Blood samples were collected; haematological and serological analyses (ELISA and confirmatory WB) were performed (Fernandez-Garcia et al., 2009). Histology and molecular (endpoint ITS-1 PCR (Cortes et al., 2007) and sequencing) analyses of tissues from a slaughtered cow with chronic besnoitiosis were carried out. Out of 59 ELISA-positive animals, 50 (23%) were confirmed by WB. B. besnoiti prevalence was higher in cows (41%) than in calves (12%); any heifer did not result positive. Considering haematological parameters, a significant shift in the differential leucocyte formula from lymphocyte to granulocyte was recorded in infected cows (Mean±S.D.:L=46.1±18.4,G=53.9±18.4) if compared to negative animals (Student’s T-test,p=0.012). This finding could be helpful in diagnosis, treatment and control of besnoitiosis. Histology revealed a high load of B. besnoiti tissue cysts in skin, vulva, muzzle, sclera, eyelid, respiratory tract, emphasizing the possibility of parasite transmission through direct contact among animals. B. besnoiti was confirmed by PCR in other organs (heart, liver, aorta wall, tonsil) and especially in ovary, uterus and vulva, suggesting that the infection could affect cows’ fertility. Parasite DNA was also found in masseters posing an important question for food security, even if B. besnoiti is not considered zoonotic. The study suggests that to investigate the dynamics of bovine besnoitiosis is mandatory associate clinical and laboratory tests, including the genetic characterization of the parasite and its eventual correlation with the disease outcome

Segmental Maternal UPD of Chromosome 7q in a Patient With Pendred and Silver Russell Syndromes-Like Features

Pendred syndrome (PS) is an autosomal recessive disorder due to mutations in the SLC26A4 gene (chr7q22. 3) and characterized by sensorineural hearing loss and variable thyroid phenotype. Silver-Russell syndrome (SRS) is a heterogeneous imprinting disorder including severe intrauterine and postnatal growth retardation, and dysmorphic features. Maternal uniparental disomy of either the whole chromosome 7 (upd(7)mat) or 7q (upd(7q)mat) is one of the multiple mechanisms impacting the expression of imprinted genes in SRS, and is associated with milder clinical features. Here, we report genetic and clinical characterization of a female child with PS, postnatal growth retardation, and minor dysmorphic features. A gross homozygous deletion of SLC26A4 exons 17-20 was suspected by Sanger sequencing and then confirmed by array-CGH. Moreover, an insertion of about 1 kb of the CCDC126 gene (7p15.3), which does not appear to be clinically relevant, was detected. The possible occurrence of a balanced rearrangement between 7p and 7q was excluded. The absence of the deletion in the father led to the investigation of upd, and microsatellite segregation analysis revealed a segmental 7q (upd(7q)mat), leading to SLC26A4 homozygosity and responsible for both PS and SRS-like traits. The proband matched 3 out of 6 major SRS criteria. In conclusion, this is the first report of uniparental isodisomy encompassing almost the whole long arm of chromosome 7 resulting in PS and SRS-like features. Whereas, the inner ear phenotype of PS is typical, the clinical features suggestive of SRS might have been overlooked

The adhesion molecule L1 regulates transendothelial migration and trafficking of dendritic cells

The adhesion molecule L1, which is extensively characterized in the nervous system, is also expressed in dendritic cells (DCs), but its function there has remained elusive. To address this issue, we ablated L1 expression in DCs of conditional knockout mice. L1-deficient DCs were impaired in adhesion to and transmigration through monolayers of either lymphatic or blood vessel endothelial cells, implicating L1 in transendothelial migration of DCs. In agreement with these findings, L1 was expressed in cutaneous DCs that migrated to draining lymph nodes, and its ablation reduced DC trafficking in vivo. Within the skin, L1 was found in Langerhans cells but not in dermal DCs, and L1 deficiency impaired Langerhans cell migration. Under inflammatory conditions, L1 also became expressed in vascular endothelium and enhanced transmigration of DCs, likely through L1 homophilic interactions. Our results implicate L1 in the regulation of DC trafficking and shed light on novel mechanisms underlying transendothelial migration of DCs. These observations might offer novel therapeutic perspectives for the treatment of certain immunological disorders

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

MALDI-TOF: Introduction in routine of a system for the rapid identification of microorganisms

Introduction. MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time of Flight) is an recently introduced technique for the rapid identification of microorganisms by mass spectrometry (1,3) (Fig.1).The aim of this work was the evaluation and validation of the MALDI-TOF method (Bruker Daltonics) for inclusion in the routine workflow of a clinical microbiology laboratory. Methods.We analyzed strains isolated from different clinical specimens. For each species of microorganism we calculated the percentage of agreement by comparing the identification of MALDI-TOF with that obtained using the methods currently in use in the laboratory. In case of discordant results the conclusive identification was carried out by sequencing of 16S ribosomal DNA (Tab.3) using the MicroSeq identification system 500 (Applied Biosystems) (2). Alpha-hemolytic streptococci were instead identified by the ATB galleries (BioMerieux).ATCC strains were used as Quality Control. Results. 1340 strains were analyzed, including 479 enterobacteria, 188 gram-negative bacilli, 189 staphylococci, 140 enterococci, 84 Haemophilus spp, 114 streptococci, 40 yeasts and 106 less common microorganisms. Gramnegative bacteria, enterococci, Staphylococcus aureus, beta-hemolytic streptococci and yeasts showed full agreement (Tab.1 e Tab.2). In addition, MALDI-TOF has proved a reliable method for identification of fastidious germs such as Legionella, Branhamella, Neisseriae, Listeria monocytogenes, Corynebacteria, and anaerobes.Alpha-hemolytic streptococci were but in most cases identified as S. pneumoniae. Conclusions. The identification by mass spectrometry allows to obtain reliable results in minutes for most of the organisms isolated from the routine. Considerable importance for the performance of the system plays the quality of the database in the instrument. The high percentage of concordance between identification with the standard methods and MALDI-TOF allowed the introduction of this method in routine workflow with the exception of alpha hemolytic streptococci for which the current system of identification is still in use

Clostridium difficile: 18 months surveillance at the Niguarda Hospital - Milano

Clostridium difficile is currently one of the most common cause of diarrhea in hospitalized or residents in long term care institutions patients, symptoms of infection ranging from diarrhea to pseudo-membranous colitis and toxic megacolon.The disease is due to the production of enterotoxin A, cytotoxin B or binary toxin. The emergence of hypervirulent new strains showing a tcdC regulatory gene deletion (ribotype 027) has been observed in recent years. Stool or rectal swab specimens were screened using automated real-time multiplex PCR (GeneXpert, Cellbio) to detect the presence of toxins producing (B toxin, binary toxin and tcdC gene deletion) C. difficile. All positive samples were cultured in order to isolate the causing infection strains.The binary toxin and/or tcdC deletion producing strains were genotyped using Multilocus Sequence Typing (MLST). MLST compares the intragenic sequences of seven housekeeping genes and provides a unique combination of alleles; to each combination is then assigned a Sequence Typing (ST). In the period January 2010 – June 2011, 3681 samples from 2234 patients were analyzed. Four hundred seventy-three patients (21.2%) were positive for the presence of C. difficile, 34 (7.2%) of there were also positive for binary toxin and 3 were positive for the tcdC gene deletion (suspected C. difficile NAP027).All the strains having tcdC gene deletion, showes ST3 typing by MLST method.These strains were isolated during the same period. The three patiens were epidemiologically linked: the patient A was hospitalized in the same room of patient B, while patient C was managed by the same team care of patient A.The spread ofsuch C. difficile strains is at the present very low in Italy. Our data confirm the poor prevalence of ribotype NAP027 and the usefulness of a presumptive diagnosis to implement immediately appropriate control measures

Immunohistochemical expression of SOX9 protein in immature, mature, and neoplastic canine Sertoli cells

Sex-determining region Y box9 gene (SOX9) protein plays a pivotal role in male sexual development. It regulates the transcription of the anti-Müllerian hormone gene promoting development of testis cords, multiplication, and maturation of Sertoli cells (SCs) and maintenance of spermatogenesis in adult testis. The immunohistochemical expression of SOX9 in normal testes has been reported in humans, mice, and rats. The present study aimed to investigate the expression of SOX9 in canine SCs during testicular maturation and neoplastic transformation. Canine testicular samples derived from three fetuses, four newborns, four prepubertal puppies, five adult dogs, 31 Sertoli cell tumors (SCTs) (one metastasizing), and five Leydig cell tumors (LCTs) were selected from departmental archive and tested immunohistochemically with a polyclonal antibody against SOX9 (1:150). All SCs from fetal, neonatal, and adult testes had a strong and exclusively nuclear labeling for SOX9. In SCs from prepubertal testes, SOX9 staining was highly variable with one negative sample (one of four), two samples with exclusively nuclear staining (two of four), and one with both nuclear and cytoplasmic labeling (one of four). Leydig cells (LCs) and LCTs were always negative. All 31 SCTs were positive for SOX9. The expression of SOX9 was nuclear, nuclear and cytoplasmic, and exclusively cytoplasmic in 18 of 31, 11 of 31, and two of 31 SCTs, respectively. This first report on the immunohistochemical expression of SOX9 in canine testes reports that in normal SCs from fetal, neonatal, and adult testes SOX9 labeled the nucleus, as in humans and laboratory animals. The cytoplasmic labeling observed in one prepubertal pairs of testes and in 11 SCTs could reflect SC immaturity or dedifferentiation, paralleling results observed in rat testes. The expression of SOX9 in SCs and SCTs and its absence in LCs and LCTs suggests that SOX9 is a reliable diagnostic marker for both normal and neoplastic SCs