Renal alterations in cats (Felis catus) housed in shelters and affected by systemic AA-amyloidosis: Clinicopathological data, histopathology, and ultrastructural features.

AA-amyloidosis is frequent in shelter cats, and chronic kidney disease is the foremost cause of death. The aims were to describe kidney laboratory and microscopic findings in shelter cats with AA-amyloidosis. Cats were included if kidney specimens were collected post-mortem and laboratory data were available within 6 months before death. Renal lesions were evaluated with optical and electron microscopy. Mass spectrometry was used to characterize amyloid. Nine domestic short-hair cats were included; 4 females and 5 males with a median age of 8 years (range = 2-13). All cats had blood analyses and urinalyses available. Serum creatinine concentrations were increased in 6 cats and symmetric dimethylarginine was increased in all of the cats. All of the cats had proteinuria. Eight of 9 cats had amyloid in the medulla, and 9 had amyloid in the cortex (glomeruli). All cats had amyloid in the interstitium. Six cats had concurrent interstitial nephritis and 1 had membranoproliferative glomerulonephritis. All cats had extrarenal amyloid deposits. Amyloid was AA in each case. In conclusion, renal deposition of amyloid occurs in both cortex and medulla in shelter cats and is associated with azotemia and proteinuria. Renal involvement of systemic AA-amyloidosis should be considered in shelter cats with chronic kidney disease. The cat represents a natural model of renal AA-amyloidosis.

Antileishmanial and Antifungal Activities of Volatile Oils from Cinnamomum cassia Bark and Schinus molle Leaves.

This study reports on the chemical composition and antileishmanial and anticandidal activities of volatile oils of Schinus molle dried leaves (SM), Cinnamomum cassia branch bark (CC) and their blends. Major constituents of SM were spathulenol (26.93%), ß-caryophyllene (19.90%), and caryophyllene oxide (12.69%), whereas (E)-cinnamaldehyde (60.11%), cinnamyl acetate (20.90%) and cis-2-methoxycinnamic acid (10.37%) were predominant in CC. SM (IC50 = 21.45 µg/mL) and CC (IC50 = 23.27 µg/mL) displayed good activity against L. amazonensis promastigotes, besides having good or moderate activity against nine Candida strains, with Minimum Inhibitory Concentration (MIC) values ranging from 31.25 to 250 µg/mL. While the three SM and CC blends were not more active than the EOs tested individually, they exhibited remarkably high antileishmanial activity, with IC50 values ranging between 3.12 and 7.04 µg/mL, which is very similar to the IC50 of amphotericin B (positive control).

Four types of scrapie in goats differentiated from each other and bovine spongiform encephalopathy by biochemical methods.

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrPSc) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrPSc (PrPres) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants-CS-1 and CS-2 (mainly Italy)-which differed in proteolytic resistance of the PrPres N-terminus. Suitable PrPres markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters.

Low fraction of the 222K PrP variant in the protease-resistant moiety of PrPres in heterozygous scrapie positive goats.

The presence of lysine (K) at codon 222 has been associated with resistance to classical scrapie in goats, but few scrapie cases have been identified in 222Q/K animals. To investigate the contribution of the 222K variant to PrPres formation in natural and experimental Q/K scrapie cases, we applied an immunoblotting method based on the use of two different monoclonal antibodies, F99/97.6.1 and SAF84, chosen for their different affinities to 222K and 222Q PrP variants. Our finding that PrPres seems to be formed nearly totally by the 222Q variant provides evidence that the 222K PrP variant confers resistance to conversion to PrPres formation and reinforces the view that this mutation has a protective role against classical scrapie in goats.

Detection of cellular prion protein in exosomes derived from ovine plasma.

Prion protein (PrP) is present at extremely low levels in the blood of animals and its detection is complicated by the poor sensitivity of current standard methodologies. Interesting results have been obtained with recent advanced technologies that are able to detect minute amounts of the pathological PrP (PrPSc), but their efficiency is reduced by various factors present in blood. In this study, we were able to extract cellular PrP (PrPC) from plasma-derived exosomes by a simple, fast method without the use of differential ultracentrifugation and to visualize it by Western blotting, reducing the presence of most plasma proteins. This result confirms that blood is capable of releasing PrP in association with exosomes and could be useful to better study its role in the pathogenesis of transmissible spongiform encephalopathies.

Detection and discrimination of classical and atypical L-type bovine spongiform encephalopathy by real-time quaking-induced conversion.

Statutory surveillance of bovine spongiform encephalopathy (BSE) indicates that cattle are susceptible to both classical BSE (C-BSE) and atypical forms of BSE. Atypical forms of BSE appear to be sporadic and thus may never be eradicated. A major challenge for prion surveillance is the lack of sufficiently practical and sensitive tests for routine BSE detection and strain discrimination. The real-time quaking-induced conversion (RT-QuIC) test, which is based on prion-seeded fibrillization of recombinant prion protein (rPrPSen), is known to be highly specific and sensitive for the detection of multiple human and animal prion diseases but not BSE. Here, we tested brain tissue from cattle affected by C-BSE and atypical L-type bovine spongiform encephalopathy (L-type BSE or L-BSE) with the RT-QuIC assay and found that both BSE forms can be detected and distinguished using particular rPrPSen substrates. Specifically, L-BSE was detected using multiple rPrPSen substrates, while C-BSE was much more selective. This substrate-based approach suggests a diagnostic strategy for specific, sensitive, and rapid detection and discrimination of at least some BSE forms.

Regional Targeted Subcutaneous Injection of Botulinum Neurotoxin Type A in Refractory Chronic Migraine: A Randomized, Double-Blind, Placebo-Controlled Study.

In this randomized, double-blind, placebo-controlled study, we evaluated the efficacy of an individualized technique of subcutaneous injection of botulinum toxin type A (BoNT-A) targeted (SjBoT) to the occipital or trigeminal skin area in non-responder patients with chronic migraine (CM). Patients who had not previously responded to at least two treatments of intramuscular injections of BoNT-A were randomly assigned (2:1) to receive two subcutaneous administrations of BoNT-A (up to 200 units) with the SjBoT injection paradigm or placebo. Following the skin area where the maximum pain began, treatment was given in the trigeminal or occipital region bilaterally. The primary endpoint changed in monthly headache days from baseline to the last 4 weeks. Among 139 randomized patients, 90 received BoNT-A and 49 received placebo, and 128 completed the double-blind phase. BoNT-A significantly reduced monthly headache days versus placebo (-13.2 versus -1.2; p < 0.0001) in the majority of patients who had cutaneous allodynia. Other secondary endpoints, including measures for disability (Migraine Disability Assessment questionnaire from baseline 21.96 to 7.59 after treatment, p = 0.028), also differed. Thus, in non-responder patients with CM, BoNT-A significantly reduced migraine days when administered according to the "follow the origin of maximum pain" approach using SjBoT injection paradigm.

Are rapid tests and confirmatory western blot used for cattle and small ruminants TSEs reliable tools for the diagnosis of Chronic Wasting Disease in Europe?

The first case of CWD in Europe was detected in a Norwegian reindeer in 2016, followed later by two CWD cases in Norwegian moose. To prevent the potential spread of CWD to the EU, the European Commission (Regulation EU 2017_1972) implemented a CWD surveillance programme in cervids in the six countries having reindeer and or moose (Estonia, Finland, Latvia, Lithuania, Poland, and Sweden). Each country had to test a minimum of 3000 cervids for CWD using diagnostic rapid tests approved by the EC Regulation. Experimental transmission studies in rodents have demonstrated that the CWD strains found in Norwegian reindeer are different from those found in moose and that these European strains are all different from the North American ones. Data on the performances of authorised rapid tests are limited for CWD (from North America) and are currently minimal for CWD from Europe, due to the paucity of positive material. The aim of this study was to evaluate the diagnostic performances of three of the so-called "rapid" tests, commercially available and approved for TSE diagnosis in cattle and small ruminants, to detect the CWD strains circulating in Europe. The performances of these three tests were also compared to two different confirmatory western blot methods. Using parallel testing on the same panel of available samples, we evaluated here the analytical sensitivity of these methods for TSE diagnosis of CWD in Norwegian cervids tissues. Our results show that all the methods applied were able to detect the CWD positive samples even if differences in analytical sensitivity were clearly observed. Although this study could not assess the test accuracy, due to the small number of samples available, it is conceivable that the rapid and confirmatory diagnostic systems applied for CWD surveillance in Northern Europe are reliable tools.

AA-amyloidosis in cats (Felis catus) housed in shelters.

Systemic AA-amyloidosis is a protein-misfolding disease characterized by fibril deposition of serum amyloid-A protein (SAA) in several organs in humans and many animal species. Fibril deposits originate from abnormally high serum levels of SAA during chronic inflammation. A high prevalence of AA-amyloidosis has been reported in captive cheetahs and a horizontal transmission has been proposed. In domestic cats, AA-amyloidosis has been mainly described in predisposed breeds but only rarely reported in domestic short-hair cats. Aims of the study were to determine AA-amyloidosis prevalence in dead shelter cats. Liver, kidney, spleen and bile were collected at death in cats from 3 shelters. AA-amyloidosis was scored. Shedding of amyloid fibrils was investigated with western blot in bile and scored. Descriptive statistics were calculated. In the three shelters investigated, prevalence of AA-amyloidosis was 57.1% (16/28 cats), 73.0% (19/26) and 52.0% (13/25), respectively. In 72.9% of cats (35 in total) three organs were affected concurrently. Histopathology and immunofluorescence of post-mortem extracted deposits identified SAA as the major protein source. The duration of stay in the shelters was positively associated with a histological score of AA-amyloidosis (B = 0.026, CI95% = 0.007-0.046; p = 0.010). AA-amyloidosis was very frequent in shelter cats. Presence of SAA fragments in bile secretions raises the possibility of fecal-oral transmission of the disease. In conclusion, AA-amyloidosis was very frequent in shelter cats and those staying longer had more deposits. The cat may represent a natural model of AA-amyloidosis.

Resistance to classical scrapie in experimentally challenged goats carrying mutation K222 of the prion protein gene.

Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP). Breeding programs have been implemented to increase scrapie resistance in sheep populations; though desirable, a similar approach has not yet been applied in goats. European studies have now suggested that several polymorphisms can modulate scrapie susceptibility in goats: in particular, PRNP variant K222 has been associated with resistance in case-control studies in Italy, France and Greece. In this study we investigated the resistance conferred by this variant using a natural Italian goat scrapie isolate to intracerebrally challenge five goats carrying genotype Q/Q 222 (wild type) and five goats carrying genotype Q/K 222. By the end of the study, all five Q/Q 222 goats had died of scrapie after a mean incubation period of 19 months; one of the five Q/K 222 goats died after 24 months, while the other four were alive and apparently healthy up to the end of the study at 4.5 years post-challenge. All five of these animals were found to be scrapie negative. Statistical analysis showed that the probability of survival of the Q/K 222 goats versus the Q/Q 222 goats was significantly higher (p = 0.002). Our study shows that PRNP gene mutation K222 is strongly associated with resistance to classical scrapie also in experimental conditions, making it a potentially positive target for selection in the frame of breeding programs for resistance to classical scrapie in goats.

Toward Linguistic Recognition of Generalized Anxiety Disorder.

Background: Generalized anxiety disorder (GAD) refers to extreme, uncontrollable, and persistent worry and anxiety. The disorder is known to affect the social functioning and well-being of millions of people, but despite its prevalence and burden to society, it has proven difficult to identify unique behavioral markers. Interestingly, the worrying behavior observed in GAD is argued to stem from a verbal linguistic process. Therefore, the aim of the present study was to investigate if GAD can be predicted from the language people use to put their anxious worries into words. Given the importance of avoidance sensitivity (a higher likelihood to respond anxiously to novel or unexpected triggers) in GAD, this study also explored if prediction accuracy increases when individual differences in behavioral avoidance and approach sensitivity are taken into account. Method: An expressive writing exercise was used to explore whether GAD can be predicted from linguistic characteristics of written narratives. Specifically, 144 undergraduate student participants were asked to recall an anxious experience during their university life, and describe this experience in written form. Clinically validated behavioral measures for GAD and self-reported sensitivity in behavioral avoidance/inhibition (BIS) and behavioral approach (BAS), were collected. A set of classification experiments was performed to evaluate GAD predictability based on linguistic features, BIS/BAS scores, and a concatenation of the two. Results: The classification results show that GAD can, indeed, be successfully predicted from anxiety-focused written narratives. Prediction accuracy increased when differences in BIS and BAS were included, which suggests that, under those conditions, negatively valenced emotion words and words relating to social processes could be sufficient for recognition of GAD. Conclusions: Undergraduate students with a high GAD score can be identified based on their written recollection of an anxious experience during university life. This insight is an important first step toward development of text-based digital health applications and technologies aimed at remote screening for GAD. Future work should investigate the extent to which these results uniquely apply to university campus populations or generalize to other demographics.

Validation of serum paraoxonase/arylesterase 1 (PON1) as a protein marker of illicit dexamethasone treatment in veal calves.

The illicit use of dexamethasone and other glucocorticoids for cattle fattening in livestock production has been widely described; evidence for illegal treatments can be obtained by direct or indirect detection. In our previous study, we applied two-dimensional electrophoresis (2DE) to identify plasma protein markers of dexamethasone administration in veal calves. Comparison of 2DE maps obtained from blood samples before and after treatment showed the disappearance of two protein spots identified as serum paraoxonase/arylesterase 1 precursor (PON1). In the present study, we validated PON1 as a marker by analysing a larger number of samples treated with dexamethasone for illicit use. Analysis of samples from experimental treatment with other glucocorticoids, androgens and oestrogens confirmed that their influence on PON1 could be excluded. The specificity of the PON1 protein marker was verified on expected negative field samples to exclude interfering factors. However, there is poor statistical evidence to support a significant association between the outcome of PON1 and the considered variables. The results on field samples were compared with histological examination of the thymus as a biomarker of corticosteroid treatment monitored in the Italian histological plan for the control of growth promoters in animals. Two suspect cases were identified from two Piedmont farms where other animals had tested positive at histological examination. In conclusion, the absence of PON1 in the plasma of veal calves can indirectly reveal illicit dexamethasone treatment in individual animals and so identify suspect farms for further investigation. It is effective in a period ranging from 3 to about 10 days from illicit treatment, covering a time span that goes beyond the limits of official chemical controls and preceding histological controls on the thymus of slaughtered animals. PON1 detection in plasma can be coupled with other tests to identify illegal dexamethasone use on veal calf farms.

Cryo-EM structure of ex vivo fibrils associated with extreme AA amyloidosis prevalence in a cat shelter.

AA amyloidosis is a systemic disease characterized by deposition of misfolded serum amyloid A protein (SAA) into cross-ß amyloid in multiple organs in humans and animals. AA amyloidosis occurs at high SAA serum levels during chronic inflammation. Prion-like transmission was reported as possible cause of extreme AA amyloidosis prevalence in captive animals, e.g. 70% in cheetah and 57-73% in domestic short hair (DSH) cats kept in zoos and shelters, respectively. Herein, we present the 3.3 Å cryo-EM structure of AA amyloid extracted post-mortem from the kidney of a DSH cat with renal failure, deceased in a shelter with extreme disease prevalence. The structure reveals a cross-ß architecture assembled from two 76-residue long proto-filaments. Despite >70% sequence homology to mouse and human SAA, the cat SAA variant adopts a distinct amyloid fold. Inclusion of an eight-residue insert unique to feline SAA contributes to increased amyloid stability. The presented feline AA amyloid structure is fully compatible with the 99% identical amino acid sequence of amyloid fragments of captive cheetah.

Emerging Treatment Options for Multi-Drug-Resistant Bacterial Infections.

Antimicrobial resistance (AMR) remains one of the top public health issues of global concern. Among the most important strategies for AMR control there is the correct and appropriate use of antibiotics, including those available for the treatment of AMR pathogens. In this article, after briefly reviewing the most important and clinically relevant multi-drug-resistant bacteria and their main resistance mechanisms, we describe the emerging antimicrobial options for both MDR Gram-positive cocci and Gram-negative bacilli, including recently marketed agents, molecules just approved or under evaluation and rediscovered older antibiotics that have regained importance due to their antimicrobial spectrum. Specifically, emerging options for Gram-positive cocci we reviewed include ceftaroline, ceftobiprole, tedizolid, dalbavancin, and fosfomycin. Emerging treatment options for Gram-negative bacilli we considered comprise ceftolozane-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, aztreonam-avibactam, minocycline, fosfomycin, eravacycline, plazomicin, and cefiderocol. An exciting scenario is opening today with the long awaited growing availability of novel molecules for the treatment of AMR bacteria. Knowledge of mechanisms of action and resistance patterns allows physicians to increasingly drive antimicrobial treatment towards a precision medicine approach. Strict adherence to antimicrobial stewardship practices will allow us to preserve the emerging antimicrobials for our future.

Wild or Farmed Gilthead Seabream (Sparus aurata)? How To Distinguish between Them by Two-Dimensional Gel Electrophoresis.

ABSTRACT: Because the world's wild fish stocks are limited and the market demand is increasing, fish farming has become an alternative food source and a way to reduce costs for consumers. The sale of farmed as wild fish is a fraudulent practice; it is, therefore, important to find new and alternative tools that can help in the fight against fraud to protect consumers and to ensure food traceability. The proteomic profiles of farmed and wild fish differ. With this study we wanted to identify liver protein markers via two-dimensional electrophoresis that would allow us to distinguish wild from farmed gilthead seabream. The liver samples from 32 gilthead seabream, wild and farmed, were stored at -80°C before protein extraction. The samples were subjected to two-dimensional electrophoresis to detect qualitative and quantitative differences. Proteomic analysis showed a protein spot (molecular weight of ∼34 kDa and isoelectric point of ∼6.9) only in the samples from the wild gilthead seabream; liquid chromatography-tandem mass spectrometry identified the spot as ubiquitin. Ubiquitin could be a valid marker to differentiate wild from farmed gilthead seabream; it could be used to ensure continuous monitoring throughout the entire commercial chain and to fight commercial fraud.

RT-QuIC detection of pathological prion protein in subclinical goats following experimental oral transmission of L-type BSE.

OBJECTIVE: The spread of bovine spongiform encephalopathy (BSE) agent to small ruminants is still a major issue in the surveillance of transmissible spongiform encephalopathies (TSEs). L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE with an unknown zoonotic potential that is transmissible to cattle and small ruminants. Our current knowledge of bovine atypical prion strains in sheep and goat relies only on experimental transmission studies by intracranial inoculation. To assess oral susceptibility of goats to L-BSE, we orally inoculated five goats with cattle L-BSE brain homogenates and investigated pathogenic prion protein (PrPsc) distribution by an ultrasensitive in vitro conversion assay known as Real-Time Quaking Induced Conversion (RT-QuIC). RESULTS: Despite a prolonged observation period of 80 months, all these animals and the uninfected controls did not develop clinical signs referable to TSEs and tested negative by standard diagnostics. Otherwise, RT-QuIC analysis showed seeding activity in five out of five examined brain samples. PrPsc accumulation was also detected in spinal cord and lymphoreticular system. These results indicate that caprine species are susceptible to L-BSE by oral transmission and that ultrasensitive prion tests deserve consideration to improve the potential of current surveillance systems against otherwise undetectable forms of animal prion infections.

Olfactory system involvement in natural scrapie disease.

The olfactory system (OS) is involved in many infectious and neurodegenerative diseases, both human and animal, and it has recently been investigated in regard to transmissible spongiform encephalopathies. Previous assessments of nasal mucosa infection by prions following intracerebral challenge suggested a potential centrifugal spread along the olfactory nerve fibers of the pathological prion protein (PrP(Sc)). Whether the nasal cavity may be a route for centripetal prion infection to the brain has also been experimentally studied. With the present study, we wanted to determine whether prion deposition in the OS occurs also under field conditions and what type of anatomical localization PrP(Sc) might display there. We report here on detection by different techniques of PrP(Sc) in the nasal mucosa and in the OS-related brain areas of sheep affected by natural scrapie. PrP(Sc) was detected in the perineurium of the olfactory nerve bundles in the medial nasal concha and in nasal-associated lymphoid tissue. Olfactory receptor neurons did not show PrP(Sc) immunostaining. PrP(Sc) deposition was found in the brain areas of olfactory fiber projection, chiefly in the olfactory bulb and the olfactory cortex. The prevalent PrP(Sc) deposition patterns were subependymal, perivascular, and submeningeal. This finding, together with the discovery of an intense PrP(Sc) immunostaining in the meningeal layer of the olfactory nerve perineurium, at the border with the subdural space extension surrounding the nerve rootlets, strongly suggests a probable role of cerebrospinal fluid in conveying prion infectivity to the nasal submucosa.

Real-life experience with ceftobiprole in a tertiary-care hospital.

OBJECTIVES: Ceftobiprole is a new therapeutic option for bacterial pneumonia, with activity against most antimicrobial-resistant Gram-positive cocci, including methicillin-resistant Staphylococcus aureus. Data on the use of ceftobiprole in real life are limited. We evaluated the efficacy and safety of ceftobiprole in a context of a hospital practice. METHODS: In a single-centre, observational, retrospective clinical study, we collected data of 29 patients undergoing ceftobiprole therapy, with a focus on clinical outcomes and adverse events. RESULTS: There was a high burden of comorbidities in the study cohort, including kidney dysfunction (38%) and cancer (24%), and a high proportion of patients with sepsis/septic shock (72%), a central line (41%) or on mechanical ventilation (21%). Most infections were nosocomial (24, 82.8%). Ceftobiprole was mostly prescribed for pneumonia (17 patients, 58.6%), and bloodstream infections (10 patients, 34.5%), both empirically (9 cases, 31%) and as targeted therapy (20, 69%, with staphylococci as the dominant pathogens). It was the first-line drug in 15 cases (51.7%). Overall, a favourable clinical outcome was observed in the majority of cases (68.9%), with clinical cure in 3 (10.3%) and clinical improvement in 17 (58.6%). Failure of treatment occurred in seven cases (24.1%). Three patients experienced a definite ceftobiprole-related adverse event, with two cases of myoclonus. No major adverse effect on bone marrow, kidney or liver function was observed. CONCLUSIONS: Ceftobiprole, even outside current indications, may be a safe and effective treatment for resistant Gram-positive cocci infections where other drugs are inactive or poorly tolerated, and for salvage therapy.

Development of a Novel Method for Rapid Discrimination between Wild and Farmed Sea Bream.

A simple method based on direct sampling analysis, coupled with a time of flight mass spectrometer, was developed to discriminate between wild and farmed sea bream on the basis of the docosahexaenoic and arachidonic fatty acid ratio. Good precision in repeatability and reproducibility (relative standard deviation < 15%) was obtained. The fatty acid ratios of the two types of fish were statistically significant (Student's t < 0.001). The use of a simple, rapid, and cost-effective tool could aid in the detection of commercial fish fraud, increase the number of controlled samples, and strengthen control along the entire commercial chain.