Development of an in Vivo Lipopolysaccharide Inflammation Model to Study the Pharmacodynamics of COX-2 Inhibitors Celecoxib, Mavacoxib, and Meloxicam in Cockatiels (Nymphicus hollandicus).

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used frequently in avian medicine for their antipyretic, analgesic, and anti-inflammatory properties during surgery and for diseases that cause tissue damage and inflammation. NSAIDs inhibit cyclooxygenase (COX) enzymes, which are responsible for the induction of pyresis, pain, and inflammation. In our study, a lipopolysaccharide-induced (LPS) pyresis model was optimized using cockatiels (Nymphicus hollandicus) as subject birds (four males/three females) and validated in two females and one male, characterized by an intravenous bolus injection of LPS (7.5 mg/kg) administered at T0 and T24 (24 hours following the first LPS injection). To demonstrate the feasibility of the model to assess pharmacodynamic (PD) parameters of different NSAIDs, mavacoxib 4 mg/kg (four males/four females), celecoxib 10 mg/kg (four males/four females) and meloxicam 1 mg/kg (four males/four females) were evaluated in the model at dosages used frequently in practice. The PD parameters (body temperature, mentation, posture, preference of location in the cage, and prostaglandin E2 [PGE2] plasma concentrations) were determined for 10 hours following the second LPS injection. At the doses evaluated, mavacoxib and celecoxib significantly reduced LPS-induced hypothermia, but had no clear effects on other clinical signs of illness. In contrast, no effect on hypothermia or clinical appearance was observed in the LPS-challenged cockatiels treated with meloxicam. All three NSAIDs were able to inhibit the increase in LPS-induced PGE2 plasma concentrations, yet the effect was most pronounced in the birds treated with meloxicam. Consequently, the presented model opens perspectives for future dose-effect PD studies to optimize analgesic protocols in cockatiels.

Subtherapeutic tetracycline concentrations aggravate Salmonella Typhimurium infection by increasing bacterial virulence.

OBJECTIVES: Antibiotics are among the most frequently prescribed drugs in human and animal medicine. With antibiotic resistance being a serious threat to veterinary and public health, the prudent use of antibiotics receives much attention. Less well known is that incorrect use of antimicrobial agents may also lead to increased bacterial virulence with the potential of a more severe clinical course of infection. Therefore, the aim of this study was to investigate the effect of subtherapeutic doses of tetracyclines on htpG virulence gene expression in Salmonella Typhimurium and on the course of salmonellosis. METHODS: Salmonella strains containing an htpG-luxCDABE transcriptional fusion were constructed. Phenotype microarrays and tetracycline treatment were used to investigate their htpG expression. A Salmonella transposon mutant bank was used to identify genes involved in the induction of htpG gene expression. Finally, the in vitro results were linked to the in vivo situation using a Salmonella mouse model. RESULTS: We demonstrate that subtherapeutic antimicrobial concentrations can exacerbate bacterial infections through direct up-regulation of bacterial virulence factors using Salmonella Typhimurium 112910a phage type 120/ad as a model organism. Phenotype microarrays showed that expression of the Salmonella Typhimurium virulence gene htpG is increased by several tetracycline antimicrobials at values below their MIC, a process that requires intact Salmonella LPS genes. Exposure of experimentally infected DBA/2J mice to subtherapeutic doxycycline concentrations resulted in htpG-mediated exacerbation of Salmonella Typhimurium infection. CONCLUSIONS: These findings show that the Salmonella isolate used in this study can respond to subtherapeutic tetracycline pressure by increasing its virulence and disease severity.

Marek's disease virus associated ocular lymphoma in Roulroul partridges (Rollulus rouloul).

Two 1-year old Roulroul partridges (Rollulus rouloul), one male and one female, were presented because of eye problems and anorexia. Twenty of the 30 Roulroul partridges in the owner's collection had already died. The affected birds stopped eating, became thinner, and eventually died. Antibiotic treatment, which started because of the suspicion of a septicaemic process, was unsuccessful. At clinical examination of the two partridges it was found that in both birds, one eye ball was filled with a whitish yellow amorphous material and the other eye ball of the female showed a distinct corneal opacity. Both presented birds were euthanized. Necropsy revealed no significant abnormalities in addition to the eye lesions. Histology and immunohistochemistry of the female's eye revealed an infiltrate of T-lymphocytes corresponding to ocular lymphoma. Herpesvirus genus-specific PCR, followed by Sanger sequencing confirmed the presumptive diagnosis of Marek's disease in both birds. To our knowledge, this is the first confirmed case of infection with Gallid Herpesvirus 2 (Marek's disease virus) in partridges and the first case in this specific species.

Pulmonary Aspergillosis in Humboldt Penguins-Susceptibility Patterns and Molecular Epidemiology of Clinical and Environmental Aspergillus fumigatus Isolates from a Belgian Zoo, 2017-2022.

Aspergillus fumigatus is the main causative agent of avian aspergillosis and results in significant health problems in birds, especially those living in captivity. The fungal contamination by A. fumigatus in the environment of Humboldt penguins (Spheniscus humboldti), located in a Belgian zoo, was assessed through the analysis of air, water, sand and nest samples during four non-consecutive days in 2021-2022. From these samples, potential azole-resistant A. fumigatus (ARAF) isolates were detected using a selective culture medium. A total of 28 veterinary isolates obtained after necropsy of Humboldt penguins and other avian species from the zoo were also included. All veterinary and suspected ARAF isolates from the environment were characterized for their azole-resistance profile by broth microdilution. Isolates displaying phenotypic resistance against at least one medical azole were systematically screened for mutations in the cyp51A gene. A total of 14 (13.6%) ARAF isolates were identified from the environment (n = 8) and from Humboldt penguins (n = 6). The TR34/L98H mutation was observed in all resistant environmental strains, and in two resistant veterinary strains. To the best of our knowledge, this is the first description of this mutation in A. fumigatus isolates from Humboldt penguins. During the period 2017-2022, pulmonary aspergillosis was confirmed in 51 necropsied penguins, which reflects a death rate due to aspergillosis of 68.0%, mostly affecting adults. Microsatellite polymorphism analysis revealed a high level of diversity among environmental and veterinary A. fumigatus isolates. However, a cluster was observed between one veterinary isolate and six environmental strains, all resistant to medical azoles. In conclusion, the environment of the Humboldt penguins is a potential contamination source of ARAF, making their management even more complex.

Identification of fungal isolates by MALDI-TOF mass spectrometry in veterinary practice: validation of a web application.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable method to identify fungal isolates. The success of this approach relies on the availability of exhaustive databases, but the latter were built with a focus on human pathogens. We assessed a large in-house database of reference spectra and a dedicated web application for their suitability for use in veterinary laboratories. A panel of 290 mold and yeast isolates representing 69 different fungal species was isolated from various animals (including pets, cattle, and zoo animals) and identified using both MALDI-TOF MS and conventional techniques. The performance of the 2 methods was compared, and identifications were confirmed by DNA sequencing. MALDI-TOF MS allowed distinction between some closely related species and achieved 89% correct identification at the species level. In comparison, only 60% of the isolates were correctly identified with conventional approaches. Using this online application, MALDI-TOF MS thus appears to be a relevant alternative for the identification of fungal isolates encountered by animal health professionals.

Comparative population pharmacokinetics and absolute oral bioavailability of COX-2 selective inhibitors celecoxib, mavacoxib and meloxicam in cockatiels (Nymphicus hollandicus).

Selective COX-2 inhibitors are non-steroidal anti-inflammatory drugs which directly target cyclooxygenase-2 (COX-2), an enzyme mainly responsible for induction of inflammation, pyresis and pain. Although commonly used in avian medicine, limited pharmacokinetic (PK) data in domestic and companion birds are available. In this study, PK parameters and absolute oral bioavailability expressed as percentage (F%) of celecoxib (10 mg/kg BW), mavacoxib (4 mg/kg BW) and meloxicam (1 mg/kg BW) were determined following single oral (PO) and intravenous (IV) administration to cockatiels (Nymphicus hollandicus). The drugs were quantified in plasma by liquid chromatography-tandem mass spectrometry. Data were processed using the nonlinear mixed effects (NLME) approach. In contrast to celecoxib (T1/2el = 0.88 h) and meloxicam (T1/2el = 0.90 h), mavacoxib has a prolonged elimination half-life (T1/2el = 135 h) following oral administration of a commercial formulation (CF). High to complete oral absorption was observed following oral administration of celecoxib (F% = 56-110%) and mavacoxib (F% = 111-113%), CF and standard solutions, respectively. In contrast, the F% of meloxicam was low (F% = 11%). Based on the presented results, a less frequent dosing of mavacoxib is proposed compared to celecoxib and meloxicam. However, pharmacodynamic and safety studies are necessary to further investigate the use of these NSAIDs in cockatiels.

Effects of urbanization on host-pathogen interactions, using Yersinia in house sparrows as a model.

Urbanization strongly affects biodiversity, altering natural communities and often leading to a reduced species richness. Yet, despite its increasingly recognized importance, how urbanization impacts on the health of individual animals, wildlife populations and on disease ecology remains poorly understood. To test whether, and how, urbanization-driven ecosystem alterations influence pathogen dynamics and avian health, we use house sparrows (Passer domesticus) and Yersinia spp. (pathogenic for passerines) as a case study. Sparrows are granivorous urban exploiters, whose western European populations have declined over the past decades, especially in highly urbanized areas. We sampled 329 house sparrows originating from 36 populations along an urbanization gradient across Flanders (Belgium), and used isolation combined with 'matrix-assisted laser desorption ionization- time of flight mass spectrometry' (MALDI-TOF MS) and PCR methods for detecting the presence of different Yersinia species. Yersinia spp. were recovered from 57.43% of the sampled house sparrows, of which 4.06%, 53.30% and 69.54% were identified as Y. pseudotuberculosis, Y. enterocolitica and other Yersinia species, respectively. Presence of Yersinia was related to the degree of urbanization, average daily temperatures and the community of granivorous birds present at sparrow capture locations. Body condition of suburban house sparrows was found to be higher compared to urban and rural house sparrows, but no relationships between sparrows' body condition and presence of Yersinia spp. were found. We conclude that two determinants of pathogen infection dynamics, body condition and pathogen occurrence, vary along an urbanization gradient, potentially mediating the impact of urbanization on avian health.

Feral pigeons: A reservoir of zoonotic Salmonella Enteritidis strains?

Salmonella enterica infections in pigeons are generally associated with pigeon-adapted strains of serovar Typhimurium that are of little public health concern. Here, we isolated Salmonella Enteritidis phage type 4 (PT4), an important human pathogen, from a population of feral pigeons in Brussels, which was further characterized by Pulsed-Field Gel Electrophoresis. All pigeon isolates belonged to the same pulsotype, which has been present in Belgian pigeons at least since 2001 and is associated with poultry and disease in humans. A high prevalence of 33% of Salmonella Enteritidis in Brussels combined with dense pigeon populations suggest that feral pigeons may constitute a significant, but unrevealed reservoir for contracting salmonellosis in the urban environment.

The Impact of Deoxynivalenol on Pigeon Health: Occurrence in Feed, Toxicokinetics and Interaction with Salmonellosis.

Seed-based pigeon diets could be expected to result in exposure of pigeons to mycotoxins such as deoxynivalenol (DON). Ingestion of low to moderate contamination levels of DON may impair intestinal health, immune function and/or pathogen fitness, resulting in altered host-pathogen interactions and thus different outcome of infections. Here we demonstrate that DON was one of the most frequently detected mycotoxins in seed-based racing pigeons feed, contaminating 5 out of 10 samples (range 177-1,466 µg/kg). Subsequently, a toxicokinetic analysis revealed a low absolute oral bioavailability (F) of DON in pigeons (30.4%), which is comparable to other avian species. Furthermore, semi-quantitative analysis using high-resolution mass spectrometry revealed that DON-3α-sulphate is the major metabolite of DON in pigeons after intravenous as well as oral administration. Following ingestion of DON contaminated feed, the intestinal epithelial cells are exposed to significant DON concentrations which eventually may affect intestinal translocation and colonization of bacteria. Feeding pigeons a DON contaminated diet resulted in an increased percentage of pigeons shedding Salmonella compared to birds fed control diet, 87 ± 17% versus 74 ± 13%, respectively. However, no impact of DON was observed on the Salmonella induced disease signs, organ lesions, faecal and organ Salmonella counts. The presented risk assessment indicates that pigeons are frequently exposed to mycotoxins such as DON, which can affect the outcome of a Salmonella infection. The increasing number of pigeons shedding Salmonella suggests that DON can promote the spread of the bacterium within pigeon populations.

House Sparrows Do Not Constitute a Significant Salmonella Typhimurium Reservoir across Urban Gradients in Flanders, Belgium.

In recent decades major declines in urban house sparrow (Passer domesticus) populations have been observed in north-western European cities, whereas suburban and rural house sparrow populations have remained relatively stable or are recovering from previous declines. Differential exposure to avian pathogens known to cause epidemics in house sparrows may in part explain this spatial pattern of declines. Here we investigate the potential effect of urbanization on the development of a bacterial pathogen reservoir in free-ranging house sparrows. This was achieved by comparing the prevalence of Salmonella enterica subspecies enterica serotype Typhimurium in 364 apparently healthy house sparrows captured in urban, suburban and rural regions across Flanders, Belgium between September 2013 and March 2014. In addition 12 dead birds, received from bird rescue centers, were necropsied. The apparent absence of Salmonella Typhimurium in fecal samples of healthy birds, and the identification of only one house sparrow seropositive for Salmonella spp., suggests that during the winter of 2013-2014 these birds did not represent any considerable Salmonella Typhimurium reservoir in Belgium and thus may be considered naïve hosts, susceptible to clinical infection. This susceptibility is demonstrated by the isolation of two different Salmonella Typhimurium strains from two of the deceased house sparrows: one DT99, typically associated with disease in pigeons, and one DT195, previously associated with a passerine decline. The apparent absence (prevalence: <1.3%) of a reservoir in healthy house sparrows and the association of infection with clinical disease suggests that the impact of Salmonella Typhimurium on house sparrows is largely driven by the risk of exogenous exposure to pathogenic Salmonella Typhimurium strains. However, no inference could be made on a causal relationship between Salmonella infection and the observed house sparrow population declines.

Host Stress Drives Salmonella Recrudescence.

Host stress is well known to result in flare-ups of many bacterial, viral and parasitic infections. The mechanism by which host stress is exploited to increase pathogen loads, is poorly understood. Here we show that Salmonella enterica subspecies enterica serovar Typhimurium employs a dedicated mechanism, driven by the scsA gene, to respond to the host stress hormone cortisol. Through this mechanism, cortisol increases Salmonella proliferation inside macrophages, resulting in increased intestinal infection loads in DBA/2J mice. ScsA directs overall Salmonella virulence gene expression under conditions that mimic the intramacrophagic environment of Salmonella, and stimulates the host cytoskeletal alterations that are required for increased Salmonella proliferation inside cortisol exposed macrophages. We thus provide evidence that in a stressed host, the complex interplay between a pathogen and its host endocrine and innate immune system increases intestinal pathogen loads to facilitate pathogen dispersal.

Genome Sequence of Devriesea agamarum, Isolated from Agamid Lizards with Dermatitis.

We report the genome sequence of Devriesea agamarum strain IMP2, isolated from the liver of a female Agama impalearis. This actinobacterium is associated with septicemia and dermatitis in agamid lizards. Availability of this genome sequence will contribute to the understanding of this pathogen's virulence.