A reverse transcription loop-mediated isothermal amplification (LAMP) assay for the detection of feline Coronavirus

Loop-mediated isothermal amplification (LAMP) is a molecular method that amplifies DNA underisothermal conditions. It relies on the use of 4 different primers recognizing 6 regions of the templatesequence and on the use of a DNA polymerase with strand displacement activity (Notomi et al., 2000).The addition of two loop primers allows the reaction time to be of one hour only (Nagamine et al., 2002).The aim of this study was to develop a reverse transcription LAMP assay for an easy and inexpensivedetection of feline Coronavirus (FCoV). Six primers binding the conserved 3’UTR region of the FCoVwere designed with the Primer Explorer software. Thirty-two samples of RNA (11 feces, 8 effusions, 9blood samples and 4 tissues) on which a reverse transcription polymerase chain reaction (RT-PCR) forthe 3’UTR region was performed were used. The reaction was carried out in 25μL reaction volume andthe mixture was incubated in a thermocycler at 63°C for 1 hour followed by 10 minutes at 80°C. LAMPproducts were visualized under UV after electrophoresis migration on a 1.5% agarose gel stained withethidium bromide, where they produce a ladder-like pattern if positive. Results where compared withthose obtained on standard PCR. Sensitivity and specificity were respectively 60% and 100% on feces,40% and 100% on effusions, 25% and 100% on blood, and 100% and 100% on tissues. The overall sensitivityand specificity of this method were of 57.1% and 100%, thus limiting a clinical application of this method,except for tissues

The gut microbiome and mucosal defenses in cats with coronaviruses: a pilot study

Feline Infectious Peritonitis (FIP) develops from a mutation of enteric feline coronaviruses (FCoVs) and an imbalance of the host immune response. The wide polymorphism of FCoVs is associated with the viral replication rate (Licitra et al. 2013).  Changes in the composition of the gut microbiota may induce quali-quantitative modifications in FCoVs and/or different immune profiles (Weese et al., 2015). Few information is available on feline gut microbiome and the association between microbiota and the predisposition to pathological conditions (Ramadan et al., 2014).The aim of this study is to provide preliminary data about the composition of gut microbiota in healthy cats compared with FCoV infected cats (with and without  FIP), in order to evaluate whether changes of gut microbiota may induce changes in FCoV, in its genetic polymorphism and in the mucosal immunity.Screening analyses have been performed on 22 cats:- Routine hematology and biochemistry on EDTA and serum (included electrophoresis and alpha-1-acid glycoprotein measurement for cats suspected with FIP)- Nested RT-PCR-3’UTR on frozen faeces- Effusion evaluation- FIV/FeLV serologyDue to strict inclusion criteria (cats younger than 2.5 years old, indoor and not assuming antibiotics in the previous two months) and based on the results obtained from the complete set of analysis, only 15 cats, specifically 5 cats for each of the following 3 groups: FIP- affected, healthy negative and positive for FCoV, have been recruited to perform the following analyses: - microbiota analysis through NGS of 16S rRNA gene (V4 region) amplicons followed by bioinformatic analysis -  evaluation of secretory IgA (ELISA kit)- phylogenetic analysis of FCoVs S gene sequencesInnovative results will be provided on the feline gut microbiota composition. These will be correlated with the presence and genetic polymorphisms of FCoV and mucosal defenses to establish significant correlations between the analysed factors

Comparison between the diagnostic accuracy of clinico-pathological and molecular tests for feline infectious peritonitis (FIP)

The aim of this study was to compare the diagnostic accuracy for feline infectious peritonitis (FIP) of conventional clinic-pathological tests with that of molecular tests such as routine PCR and PCR followed by the sequencing of the Spike (S) gene. Blood, effusion and tissues specimens were collected from 21 FIP suspected cats. In vivo examination consisted of CBC, serum protein electrophoresis, AGP measurement, cytological and biochemical examination and the evaluation of the ΔTNC on effusions, and of molecular tests such the screening PCR (target: 3’UTR region) and the PCR directed towards the S gene followed by the amplification products sequencing in order to detect the aminoacidic substitution recently considered diagnostic for FIP1. These molecular techniques were applied to tissues collected during necropsy, which also allowed forming an FIP group (13 cats) and a non-FIP group (5 cats) based on histology and immunohistochemistry. The best test on tissues was immunohistochemistry (sens: 92.3%; spec: 100%), while the screening PCR suffered of low specificity (spec: 33.3%) and the S gene sequencing showed low sensitivity (sens: 69.2%).On effusions, the best tests resulted screening PCR and cytology (sens and spec: 100%) in comparison with the ΔTNC measurement (sens: 85.7 %; spec: 100%) and the S gene sequencing (sens: 42.8%; spec: 100%).On blood, the best test resulted AGP measurement (sens: 81.8%; spec: 100%), while serum protein electrophoresis showed a surprisingly low sensitivity (sens: 41.7%). Screening PCR (sens: 55.6%; spec: 100%) and S gene sequencing (sens: 33.3%; spec: 100%) proved again low accuracy.

Isolated slaughterhouse liver as model for normothermic perfusion after warm and cold ischemia: single case report

AbstractLiver transplantation is an ultimate procedure in patients suffering end-stage liver diseases. In these last years the donation after cardiac death (DCD) has increased the pool of potential liver donors. Different studies and procedures are involved in the prevention of the main ischemic problems during the reconditioning and resuscitation of the marginal livers. Normothermic extracorporeal liver perfusion (NELP) avoids prolonged cold storage damage that is the main cause of steatosis and biliary tract ischemia in transplanted patiens. Different porcine models have been studied and developed to understand the ischemia mechanism and to select the better technique for NELP.We conducted our study using a DCD pig liver model collected from slaughterhouse. Using extracorporeal membrane oxygenation, 2000 ml of total fluid containing autologous blood, lidocaine, heparin, antibiotics, glucose 10 % solution and flunixin, the NELP was achieved. The liver was perfused over 7 hours after 48 hours of cold storage (4C°), using Eurocollins solution. During the liver withdrawal in the slaughterhouse 20 minutes were waited to simulate the warm ischemia (WI) time. Histological samples, swab for bacterial grow, blood sample, temperature and pulse oximetry saturation were collected to assess the liver viability and function. These analyses revealed stable metabolism throughout perfusion identifying a cycles 2 hours length, coinciding with recovery of oxygen uptake rates to fresh liver, as described in literature.In summary the preliminary established model of isolated hemoperfused slatherhouse liver reveals the important role of the relation between cold storage and normothermic perfusion. Moreover this preliminary study justifies further investigation of the optimization of the treatment protocols and perfusion media

Concordance between Histology, Immunohistochemistry, and RT-PCR in the Diagnosis of Feline Infectious Peritonitis

Histology, immunohistochemistry (IHC), and reverse transcription polymerase chain reaction (RT-PCR) have been used to diagnose feline infectious peritonitis (FIP), but no information regarding the comparison of their diagnostic performances on the same organ is available. The aims of this study were to determine the concordance among these tests and to evaluate which combination of tests and organs can be used in vivo. Histology, IHC, and nested RT-PCR (RT-nPCR) for feline coronavirus (FCoV) were performed on spleen, liver, mesenteric lymph node, kidney, large and small intestine, and lung from 14 FIP and 12 non-FIP cats. Sensitivity, specificity, predictive values, likelihood ratios, and concordance were calculated. IHC and RT-nPCR had the highest concordance in lung and liver, histology and IHC in the other organs. The sensitivity of histology, IHC, and RT-nPCR on the different organs ranged from 41.7 to 76.9%, 46.2 to 76.9%, and 64.3 to 85.7%, respectively, and their specificity ranged from 83.3 to 100.0%, 100% and 83.3 to 100.0%. Therefore, IHC is recommended when histology is consistent with FIP. If RT-nPCR is performed as the first diagnostic approach, results should always be confirmed with IHC. Lung or liver provide accurate information regardless of the method, while IHC is preferred to RT-nPCR to confirm FIP in the kidney or intestine

Chitinase-1 Activity in Serum of Cats with FIP

Background: Chitotriosidase (chitinase 1 or CHIT1) is secreted by activated macrophages. Macrophages are involved in the pathogenesis of feline infectious peritonitis (FIP). No reports on CHIT1 activity in cats with FIP are available. Objective: To preliminarily investigate the possible changes in serum CHIT1 activity in cats with FIP. Methods: CHIT1 activity was measured in serum samples from clinically healthy cats (n = 17), cats with FIP (n = 19) and cats with diseases potentially characterized by macrophage activation (n = 20), after a preliminary assessment of the imprecision and linearity of the method. Results: The highest CHIT1 activity was found in cats with FIP, followed by sick cats and clinically healthy cats. The magnitude of the differences between groups was higher than the intra- and inter-assay imprecision of the method (57%, respectively). Based on receiver operating characteristic (ROC) curves, CHIT1 may differentiate sick from clinically healthy cats and, to a lesser extent, cats with FIP from cats without FIP. Conclusions: CHIT1 activity may identify sick cats and, within the appropriate clinical context, cats with FIP, although larger and more standardized studies, coupled with additional information on analytical performances of the method, are required to fully explore the diagnostic or prognostic potential of this test for FIP

Chylopericardium Effusion in a Lac Alaotra Bamboo Lemur (<i>Hapalemur alaotrensis</i>)

An 11-year-old female Hapalemur alaotrensis was evaluated following a history of dyspnea of 15 days’ duration. Thoracic radiography performed by the referring veterinarian revealed a large cardiac silhouette and dorsal deviation of the trachea. Heart sounds were muffled. Echocardiographic findings were indicative of severe pericardial effusion without cardiac tamponade. No pleural effusion was identified. A computed tomography (CT) exam confirmed the presence of severe pericardial effusion and allowed identification of a parenchymatous mediastinal lesion sited at the level of the left hemithorax. To delineate the thoracic duct, lymphoCT was also performed by injection of iodinated contrast medium in the perianal subcutaneous tissue. Pericardiocentesis yielded a considerable amount of effusion with chylous biochemical and cytological properties. A diagnosis of chylopericardium with absence of pleural effusion was made. Initially, the chylopericardium was managed conservatively with two centesis and oral treatment with prednisolone. Medical treatment did not result in complete resolution of effusion and clinical signs; therefore, subtotal pericardiectomy and thoracic duct ligation were recommended. After the second pericardiocentesis, the subject died and the pericardiectomy could not be performed. To the authors’ knowledge, this is the first report of the development of chylopericardium in a Hapalemur alaotrensis

Feline morbillivirus in Northern Italy: prevalence in urine and kidneys with and without renal disease

Feline morbillivirus (FeMV) is an emerging virus that was first described in Hong Kong in 2012. Several reports suggested the epidemiological association of FeMV infection with chronic kidney disease (CKD) in cats. The aim of this study was to investigate the presence and the genetic diversity of FeMV as well as the relationship between FeMV infection and CKD in cats from Northern Italy. Urine (n\u2009=\u200981) and kidney samples (n\u2009=\u200927) from 92 cats admitted to the Veterinary Teaching Hospital of the University of Milan between 2014 and 2017 were investigated for FeMV infection. FeMV RNA was detected in one urine sample (1.23%; 95% CI: 0.03-6.68%) and in two kidneys (7.40%; 95% CI: 0.91-24.28%). FeMV RNA was revealed only in urine or kidneys of cats without evidence of CKD. Phylogenetic analysis showed that the three strains clustered with FeMV strains retrieved from public database, forming a distinct sub-cluster of FeMV. The presence of distinct genotypes of FeMV found in this study is in accordance with previous studies demonstrating that FeMV strains are genetically diverse. A clear relationship between the presence of FeMV infection and CKD in the cats from Northern Italy was not observed, confirming recent reports that do not support the hypothesis that FeMV infection is associated with the development of CK

Do Dogs and Cats Passively Carry SARS-CoV-2 on Hair and Pads?

The epidemiological role of domestic animals in the spread and transmission of SARS-CoV-2 to humans has been investigated in recent reports, but some aspects need to be further clarified. To date, only in rare cases have dogs and cats living with COVID-19 patients been found to harbour SARS-CoV-2, with no evidence of pet-to-human transmission. The aim of the present study was to verify whether dogs and cats act as passive mechanical carriers of SARS-CoV-2 when they live in close contact with COVID-19 patients. Cutaneous and interdigital swabs collected from 48 dogs and 15 cats owned by COVID-19 patients were tested for SARS-CoV-2 by qRT-PCR. The time elapsed between owner swab positivity and sample collection from pets ranged from 1 to 72 days, with a median time of 23 days for dogs and 39 days for cats. All samples tested negative, suggesting that pets do not passively carry SARS-CoV-2 on their hair and pads, and thus they likely do not play an important role in the virus transmission to humans. This data may contribute to confirming that the direct contact with the hair and pads of pets does not represent a route for the transmission of SARS-CoV-2