An extremely rare serovar of Salmonella enterica (Yopougon) discovered in a Western Whip Snake (Hierophis viridiflavus) from Montecristo Island, Italy: case report and review.

Reptiles, including snakes, can be asymptomatically infected with multiple pathogen microorganisms, including Salmonella spp., which is considered an important concern for public and animal health. Small and uninhabited isles are quite ecologically different from mainland and represent interesting fields of study, to discover unexpected biological and microbiological aspects of their wild inhabitants. This work reports the presence of the very rare Salmonella enterica serovar Yopougon, isolated in a carcass of a native wild snake (Hierophis viridiflavus) from an Italian uninhabited island of Mediterranean Sea, Montecristo. To our knowledge, S. enterica serovar Yopougon was previously isolated only once 34 years earlier in Ivory Coast, from a human fecal sample. In the present study, we present the genomic characterization of the new isolate, the phylogenetic comparison with the previously isolated S. enterica serovar Yopougon strain of human origin and with other sequences available in public databases. In addition, an extensive review of available data in the literature and from our case history is provided. Our finding represents an example of the ability of some pathogens to travel for very long distances within their hosts and then to infect others, even from different taxa.

Multivariate Genomic Hybrid Prediction with Kernels and Parental Information.

Genomic selection (GS) plays a pivotal role in hybrid prediction. It can enhance the selection of parental lines, accurately predict hybrid performance, and harness hybrid vigor. Likewise, it can optimize breeding strategies by reducing field trial requirements, expediting hybrid development, facilitating targeted trait improvement, and enhancing adaptability to diverse environments. Leveraging genomic information empowers breeders to make informed decisions and significantly improve the efficiency and success rate of hybrid breeding programs. In order to improve the genomic ability performance, we explored the incorporation of parental phenotypic information as covariates under a multi-trait framework. Approach 1, referred to as Pmean, directly utilized parental phenotypic information without any preprocessing. While approach 2, denoted as BV, replaced the direct use of phenotypic values of both parents with their respective breeding values. While an improvement in prediction performance was observed in both approaches, with a minimum 4.24% reduction in the normalized root mean square error (NRMSE), the direct incorporation of parental phenotypic information in the Pmean approach slightly outperformed the BV approach. We also compared these two approaches using linear and nonlinear kernels, but no relevant gain was observed. Finally, our results increase empirical evidence confirming that the integration of parental phenotypic information helps increase the prediction performance of hybrids.

A prospective international multi-center study on safety and efficacy of deep brain stimulation for resistant obsessive-compulsive disorder.

Deep brain stimulation (DBS) has been proposed for severe, chronic, treatment-refractory obsessive-compulsive disorder (OCD) patients. Although serious adverse events can occur, only a few studies report on the safety profile of DBS for psychiatric disorders. In a prospective, open-label, interventional multi-center study, we examined the safety and efficacy of electrical stimulation in 30 patients with DBS electrodes bilaterally implanted in the anterior limb of the internal capsule. Safety, efficacy, and functionality assessments were performed at 3, 6, and 12 months post implant. An independent Clinical Events Committee classified and coded all adverse events (AEs) according to EN ISO14155:2011. All patients experienced AEs (195 in total), with the majority of these being mild (52% of all AEs) or moderate (37%). Median time to resolution was 22 days for all AEs and the etiology with the highest AE incidence was 'programming/stimulation' (in 26 patients), followed by 'New illness, injury, condition' (13 patients) and 'pre-existing condition, worsening or exacerbation' (11 patients). Sixteen patients reported a total of 36 serious AEs (eight of them in one single patient), mainly transient anxiety and affective symptoms worsening (20 SAEs). Regarding efficacy measures, Y-BOCS reduction was 42% at 12 months and the responder rate was 60%. Improvements in GAF, CGI, and EuroQol-5D index scores were also observed. In sum, although some severe AEs occurred, most AEs were mild or moderate, transient and related to programming/stimulation and tended to resolve by adjustment of stimulation. In a severely treatment-resistant population, this open-label study supports that the potential benefits outweigh the potential risks of DBS.

Unraveling the role of male reproductive tract and haemolymph in cantharidin-exuding Lydus trimaculatus and Mylabris variabilis (Coleoptera: Meloidae): a comparative transcriptomics approach.

BACKGROUND: Meloidae (blister beetles) are known to synthetize cantharidin (CA), a toxic and defensive terpene mainly stored in male accessory glands (MAG) and emitted outward through reflex-bleeding. Recent progresses in understanding CA biosynthesis and production organ(s) in Meloidae have been made, but the way in which self-protection is achieved from the hazardous accumulation and release of CA in blister beetles has been experimentally neglected. To provide hints on this pending question, a comparative de novo assembly transcriptomic approach was performed by targeting two tissues where CA is largely accumulated and regularly circulates in Meloidae: the male reproductive tract (MRT) and the haemolymph. Differential gene expression profiles in these tissues were examined in two blister beetle species, Lydus trimaculatus (Fabricius, 1775) (tribe Lyttini) and Mylabris variabilis (Pallas, 1781) (tribe Mylabrini). Upregulated transcripts were compared between the two species to identify conserved genes possibly involved in CA detoxification and transport. RESULTS: Based on our results, we hypothesize that, to avoid auto-intoxication, ABC, MFS or other solute transporters might sequester purported glycosylated CA precursors into MAG, and lipocalins could bind CA and mitigate its reactivity when released into the haemolymph during the autohaemorrhaging response. We also found an over-representation in haemolymph of protein-domains related to coagulation and integument repairing mechanisms that likely reflects the need to limit fluid loss during reflex-bleeding. CONCLUSIONS: The de novo assembled transcriptomes of L. trimaculatus and M. variabilis here provided represent valuable genetic resources to further explore the mechanisms employed to cope with toxicity of CA in blister beetle tissues. These, if revealed, might help conceiving safe and effective drug-delivery approaches to enhance the use of CA in medicine.

Close encounters of the three morphs: Does color affect aggression in a polymorphic lizard?

Color polymorphism is genetically controlled, and the process generating and maintaining morphs can affect speciation/extinction rates. Color badges are useful signals in intraspecific communication because they convey information about alternative strategies and can potentially decrease unnecessary conflicts among different color morphs. Competition and aggressive interactions among color morphs can contribute to polymorphism maintenance. This could lead to an uneven spatial distribution of morphs in a population because the local frequency of each morph establishes the intensity of the competition and the fitness of each male. We used a polymorphic lizard, Podarcis muralis, to assess if aggression varies among morphs under two contrasting hypotheses: a heteromorphic versus homomorphic aggression. We used laboratory mirror tests after lizard color manipulation, and we verified the consistency of results with an analysis of the spatial distribution of morphs in a wild population. Both the experiments confirmed that aggression is more common during homomorphic than heteromorphic contests. The adoption of alternative behavioral strategies that minimize risks and costs could facilitate the stable coexistence of the phenotypes and reduce competition. A bias in aggression would advantage rarer morph, which would suffer less harassment by common morphs obtaining a fitness advantage. This process would be negatively-frequency-dependent and would stabilize polymorphism, possibly contributing to sympatric speciation.

Pathological findings in genital organs of bulls naturally infected with Besnoitia besnoiti.

Bulls chronically affected by bovine besnoitiosis can suffer from sterility. There is limited information about the distribution of Besnoitia cysts and their associated lesions within the male genital organs. This work describes the gross and histological abnormalities in the genital organs of 6 bulls chronically infected with Besnoitia besnoiti, including both clinically (n = 4) and subclinically (n = 2) affected cases. Parasitic cysts were observed in the genital organs of all the clinically affected bulls. The tissue cysts were most commonly found within the pampiniform plexus (4/4), where they were often seen within venous vascular walls and associated with vasculitis, followed by epididymis (3/4), tunica albuginea (2/4), and penis (1/4). In decreasing order of their frequency, observed abnormalities included seminiferous tubule degeneration, testicular fibrosis, testicular necrosis, lack of/or diminished numbers of spermatozoa, testicular atrophy, and Leydig cell hyperplasia. Only one of the subclinically infected bulls had few Besnoitia cysts within the pampinoform plexus, which was associated to small areas of necrosis and mineralization in the ipsilateral testicle. Results indicate that Besnoitia cysts and genital abnormalities are frequent in bulls chronically affected by bovine besnoitiosis, while they are mild and scarce in subclinically affected ones. Moreover, present data show that Besnotia-associated testicular lesions can occur without the presence of cysts within the testicular parenchyma. B. besnoiti cysts seem to have a tropism for the vascular structures of the spermatic chord, which may cause testicular abnormalities via vascular damage, reduced blood flow, and/or impaired thermoregulation and subsequently lead to the observed testicular lesions.

Conformational analysis of Pneumococcal Surface Antigen A (PsaA) upon zinc binding by fluorescence spectroscopy.

PsaA (pneumococcal surface antigen A) is a S. pneumoniae virulence factor that belongs to the metal transport system. The Manganese PsaA binding has been associated with oxidative stress resistance becoming a pivotal element in the bacteria virulence. It has been shown that Zinc inhibits the Manganese acquisition and promotes bacteria toxicity. We have performed a PsaA conformational analysis both in the presence (Zn-rPsaA) and in the absence of Zinc (free-rPsaA). We performed experiments in the presence of different Zinc concentrations to determine the metal minimum concentration which induced a conformational change. The protein in free and Zn-binding condition was also studied in pH ranging 2.6-8.0 and in temperature ranging 25oC-85oC. pH experiments showed a decrease of fluorescence intensity only in acidic medium. Analysis of the heat-induced denaturation demonstrated that Zinc-binding promoted an increase in melting temperature from 55oC (free-rPsaA) to 78.8oC (Zn-rPsaA) according to fluorescence measurements. In addition, the rPsaA stabilization by Zinc was verified through analysis of urea and guanidine hydrochloride denaturation. Data showed that Zinc promoted an increase in the rPsaA stability and its removal by EDTA can lead to a PsaA intermediate conformation. These findings can be considered in the development of vaccines containing PsaA as antigen.

Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system.

The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.

Experimental design approach in recombinant protein expression: determining medium composition and induction conditions for expression of pneumolysin from Streptococcus pneumoniae in Escherichia coli and preliminary purification process.

BACKGROUND: Streptococcus pneumoniae (S. pneumoniae) causes several serious diseases including pneumonia, septicemia and meningitis. The World Health Organization estimates that streptococcal pneumonia is the cause of approximately 1.9 million deaths of children under five years of age each year. The large number of serotypes underlying the disease spectrum, which would be reflected in the high production cost of a commercial vaccine effective to protect against all of them and the higher level of amino acid sequence conservation as compared to polysaccharide structure, has prompted us to attempt to use conserved proteins for the development of a simpler vaccine. One of the most prominent proteins is pneumolysin (Ply), present in almost all the serotypes known at the moment, which shows an effective protection against S. pneumoniae infections. RESULTS: We have cloned the pneumolysin gene from S. pneumoniae serotype 14 and studied the effects of eight variables related to medium composition and induction conditions on the soluble expression of rPly in Escherichia coli (E. coli) and a 28-4 factorial design was applied. Statistical analysis was carried out to compare the conditions used to evaluate the expression of soluble pneumolysin; rPly activity was evaluated by hemolytic activity assay and served as the main response to evaluate the proper protein expression and folding. The optimized conditions, validated by the use of triplicates, include growth until an absorbance of 0.8 (measured at 600 nm) with 0.1 mM IPTG during 4 h at 25°C in a 5 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl, 1 g/L glucose medium, with addition of 30 µg/mL kanamycin. CONCLUSIONS: This experimental design methodology allowed the development of an adequate process condition to attain high levels (250 mg/L) of soluble expression of functional rPly in E. coli, which should contribute to reduce operational costs. It was possible to recover the protein in its active form with 75% homogeneity.

Identification of immunodominant antigens in canine leptospirosis by Multi-Antigen Print ImmunoAssay (MAPIA).

BACKGROUND: The microscopic agglutination test (MAT), the standard method for serological diagnosis of leptospirosis, may present limitations regarding its sensitivity. Current studies suggest that Leptospira immunoglobulin-like (Lig) proteins and LipL32 are of particular interest as serodiagnostic markers since they are present only in pathogenic species of the Leptospira genus. The purpose of this study was to identify leptospiral immunodominant proteins that are recognized by canine sera from diseased dogs. RESULTS: A total of 109 dogs were studied, including seroreactive dogs (MAT ≥800) and dogs with no seroreactivity detectable by MAT. Eight recombinant fragments (31-70 kDa) of pathogenic Leptospira were tested for their use as diagnostic markers for canine leptospirosis using the Multi-antigen Print Immunoassay (MAPIA) platform: LigB [582-947aa] from L. interrogans serovar Pomona, L. interrogans serovar Copenhageni and L. kirschneri serovar Gryppotyphosa, LigB [131-649aa] from L. interrogans serovar Copenhageni, L. interrogans serovar Canicola and L. kirschneri serovar Gryppotyphosa, LigA [625-1224aa] L. interrogans serovar Copenhageni and LipL32 from L. interrogans serovar Copenhageni. The data were analyzed and ROC curves were generated. Altogether, LigB [131-649aa] L. interrogans Canicola, LigB [131-649aa] L. kirschneri Gryppotyphosa and LipL32 L. interrogans Copenhageni showed best accuracy (AUC = 0.826 to 0.869), with 70% specificity and sensitivity ranging from 89% to 95%. CONCLUSIONS: These results reinforce their potential as diagnostic candidates for the development of new methods for the serological diagnosis of canine leptospirosis.

Bioremediation potential of the Chlorella and Scenedesmus microalgae in explosives production effluents.

This study explores microalgae-based bioremediation for treating black gunpowder production effluents, an understudied yet environmentally significant stream. Two native microalgae, Chlorella sp. MC18 (CH) and Scenedesmus sp. MJ23-R (SC), were assessed for growth kinetics and nutrient removal capabilities in culture media containing different proportions of untreated raw wastewater. Results show both species thrived in 100 % raw wastewater, displaying robust growth and substantial biomass production in parallelepiped-shaped photobioreactors. SC showed superior performance, with higher maximum specific growth rate (0.549 d-1), biomass yield (454.57 mg L-1) and biomass productivity (64.94 mg L-1 d-1) compared to CH (0.524 d-1, 380.60 mg L-1, 54.37 mg L-1 d-1, respectively). The use of 100 % raw wastewater as a culture medium eliminated the need for additional freshwater input, thus reducing the water footprint. The bioremediation process also resulted in a high removal efficiency in turbidity (>95 % CH, >76 % SC), total suspended solids (>93 % CH, >74 % SC), biochemical oxygen demand (BOD5) (>62 % CH, >93 % SC) and chemical oxygen demand (COD) (>63 % CH, >87 % SC), bringing the effluent into compliance with environmental regulations. Although nitrogen (>45 % CH, >57 % SC) and sulphate (>43 % CH, >35 % SC) removal efficiencies was high, potassium bioremediation was limited (<6 %). The proximate chemical composition of the microalgal biomass revealed different allocations to carbohydrates, lipids and proteins. The results suggest promising applications for biofuel production and aquaculture. This research highlights the potential of microalgae-based bioremediation for sustainable wastewater management in the explosives industry, contributing to the UN Sustainable Development Goals and promoting green industrial practices.

Sex differences in clinical response to deep brain stimulation in resistant obsessive-compulsive disorder.

INTRODUCTION: Deep brain stimulation (DBS) is an effective alternative to treat severe refractory obsessive-compulsive disorder (OCD), although little is known on factors predicting response. The objective of this study was to explore potential sex differences in the pattern of response to DBS in OCD patients. METHODS: We conducted a prospective observational study in 25 patients with severe resistant OCD. Response to treatment was defined as a ≥35% reduction in Yale-Brown Obsessive Compulsive Scale (Y-BOCS) score. Logistic regression models were calculated to measure the likelihood of response at short and long-term follow-up by sex as measured by Y-BOCS score. Similar analyses were carried out to study changes in depressive symptomatology assessed with the Hamilton Depression Rating Scale (HDRS). Additionally, effect sizes were calculated to assess clinical significance. RESULTS: We did not observe significant clinical differences between men and women prior to DBS implantation, nor in the response after one year of stimulation. At long-term follow-up, 76.9% of men could be considered responders to DBS versus only 33.3% of women. The final response odds ratio in men was 10.05 with significant confidence intervals (88.90-1.14). No other predictors of response were identified. The sex difference in Y-BOCS reduction was clinically significant, with an effect size of 3.2. The main limitation was the small sample size. CONCLUSIONS: Our results suggest that gender could influence the long-term response to DBS in OCD, a finding that needs to be confirmed in new studies given the paucity of results on predictors of response to DBS.

Locally administered prostaglandin E2 prevents aeroallergen-induced airway sensitization in mice through immunomodulatory mechanisms.

Prostaglandin E2 attenuates airway pathology in asthmatic patients and exerts a protective effect in antigen-sensitized mice when administered systemically. We aimed to establish the consequences of intranasal PGE2 administration on airway reactivity to aeroallergens in mice and reveal the underlying immunoinflammatory mechanisms. PGE2 was administered either daily during a 10-day exposure to house dust mite (HDM) extracts or for limited intervals. Airway hyperreactivity was measured by whole-body and invasive plethysmography. The phenotypes of lung immune cells and cytokine production were analysed by flow cytometry and ELISA, respectively. Airway hyperreactivity was sustainably reduced only when PGE2 administration was restricted to the initial 5 days of exposure to HDM. Lung inflammation, IL-4 production, and airway mast cell activity were also prevented under this early short-term treatment with PGE2. Interestingly, a Th2 response was already committed on day 5 of exposure to HDM. This was paralleled by GM-CSF and osteopontin upregulation and a decreased number of plasmacytoid dendritic and T regulatory cells, as well as a trend towards reduced IL-10 expression. Local PGE2 administration prevented the increase of airway IL-13 and osteopontin and kept lung plasmacytoid dendritic cell counts close to baseline. GM-CSF and Tregs were unaffected by the treatment. These findings suggest that the protection provided by PGE2 is a result of the modulation of early lung immunomodulatory mechanisms, and possibly a shift in the balance of dendritic cells towards a tolerogenic profile.

Does the economic freedom hinder the underground economy? Evidence from a cross-country analysis.

We evaluate the impact that the economic freedom exerts on the shadow economy for a sample of 152 countries from 1995 to 2017. In order to solve endogeneity issues, we rely on an instrumental variable approach and find that a change in the economic freedom index, induced by the level of independence of financial markets from government actions, adversely affects the hidden economy. To corroborate the interpretation of our results we also show how each subcomponent of the economic freedom index explains the downward change registered in the shadow economy. Further, the negative effect is mainly found in countries characterized by a low level of democracy and strong corruption, whereas in more democratic and less corrupt countries the economic freedom positively affects the size of the shadow economy. Consistent with these findings, we also highlight that the effect of the composite indicator of economic freedom on the hidden economy is U-shaped and this relationship is exclusively driven by both business regulation and the freedom in the legal system and property rights.

Needle tract seeding and malignant transformation of hepatocellular adenoma into well-differentiated hepatocellular carcinoma in a dog.

An 11-year-old neutered female Golden Retriever was referred for investigation of marked increases in liver enzyme activities. Abdominal ultrasound revealed a large pedunculated liver mass. Diagnosis of hepatocellular adenoma (HCA) was made when the mass was excised after a first unsuccessful attempt through ultrasound-guided core-needle biopsy. One and a half years after presentation, a nodule embedded between muscles of the abdominal wall appeared. The mass was first diagnosed as a well-differentiated hepatocellular carcinoma (HCC) through cytologic examination, which was later confirmed with histopathology. Ki 67 immunostaining of the abdominal wall nodule showed an increased immunoreactivity compared with the liver mass. Therefore, the present case documents the first needle-tract seeding of a hepatocellular epithelial tumor with possible malignant transformation of HCA into a well-differentiated HCC in a dog.

Optimization of medium formulation and seed conditions for expression of mature PsaA (pneumococcal surface adhesin A) in Escherichia coli using a sequential experimental design strategy and response surface methodology.

PsaA, a candidate antigen for a vaccine against pneumonia, is well-conserved in all Streptococcus pneumoniae serotypes. A sequence of two-level experimental designs was used to evaluate medium composition and seed conditions to optimize the expression of soluble mature PsaA in E. coli. A face-centered central composite design was first used to evaluate the effects of yeast extract (5 and 23.6 g/L), tryptone (0 and 10 g/L), and glucose (1 and 10 g/L), with replicate experiments at the central point (14.3 g/L yeast extract, 5 g/L tryptone, 5.5 g/L glucose). Next, a central composite design was used to analyze the influence of NaCl concentration (0, 5, and 10 g/L) compared with potassium salts (9.4 g/L K(2)HPO(4)/2.2 g/L KH(2)PO(4)), and seed growth (7 and 16 h). Tryptone had no significant effect and was removed from the medium. Yeast extract and glucose were optimized at their intermediate concentrations, resulting in an animal-derived material-free culture medium containing 15 g/L yeast extract, 8 g/L glucose, 50 µg/mL kanamycin, and 0.4% glycerol, yielding 1 g/L rPsaA after 16 h induction at 25°C in shake flasks at 200 rpm. All the seed age and salt conditions produced similar yields, indicating that no variation had a statistically significant effect on expression. Instead of growing the seed culture for 16 h (until saturation), the process can be conducted with 7 h seed growth until the exponential phase. These results enhanced the process productivity and reduced costs, with 5 g/L NaCl being used rather than potassium salts.

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages.

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during meta-cyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

Experts' opinions about lasting innovative technologies in City Logistics.

The COVID-19 pandemic has highlighted the relevance of goods delivery in urban areas. However, this activity often generates negative environmental impact and several technologies have been proposed in recent years to reduce it, thus forming a complex innovation landscape characterized by different levels of maturity and effects on the City Logistics (CL) system. This complexity causes a deep uncertainty over the future of CL. This paper aims to tackle this uncertainty by forecasting the future of a set of CL technologies. A Delphi survey has been submitted to experts of this field to achieve a stable consensus over 33 projections related to 7 CL technologies for the year 2030. Results show that real-time data collection will help the coordination process between stakeholders, engendering an increased awareness over the value of using logistics data as well as its potential drawbacks. Moreover, experts share a positive attitude towards the expansion of Parcel Lockers, which should be monitored by public authorities to avoid a negative impact on land use. Finally, technologies such as drones and crowd-logistics have drawn the lowest level of consensus due to their lower level of maturity, which arouse the necessity to further explore several issues such as legal and technical barriers.

Outbreaks of Acute Necrotic Enteritis and Haemolytic Crisis and Renal Disease Associated with Clostridium perfringens in Captive Toucans (Ramphastos spp).

Four toco toucans (Ramphastos toco), one channel-billed toucan (Ramphastos vitellinus) and one white-throated toucan (Ramphastos tucanus) died in two disease outbreaks in the same aviary in 2011 and 2016. Post-mortem examination revealed diffuse necrotic enteritis (NE) as the cause of death of five of these six birds. Clostridium perfringens was identified by culture and real-time multiplex PCR for C. perfringens α-, ß-, ε- and ι-toxin genes in ligated intestine of one toucan from each outbreak. At another aviary, two keel-billed toucans (Ramphastos sulfuratus) died peracutely from severe haemolytic crisis with haemoglobinaemic nephrosis and cholestasis and acute tubulointerstitial nephritis. Mild NE was present in these birds and C. perfringens was demonstrated in liver by bacterial culture and real-time multiplex PCR for C. perfringens α-, ß-, ε- and ι-toxin genes. To the authors' knowledge, this is the first description of outbreaks of NE associated with C. perfringens in captive toucans. Although haemolytic crisis has been reported in humans with C. perfringens type A septicaemia and hepatic abscesses, this presentation appears not to have been described in C. perfringens infections in toucans or other avian species. The factors causing C. perfringens proliferation and disease in the toucans were not identified. PCR for C. perfringens NetB toxin and enterotoxin genes performed retrospectively on one of the C. perfringens isolates from the second outbreak and on paraffin-embedded tissues from one dead toucan from the first outbreak was negative. With the current C. perfringens toxin typing scheme, C. perfringens type A was identified in the first two outbreaks.