Addressing Challenges in Wildlife Rehabilitation: Antimicrobial-Resistant Bacteria from Wounds and Fractures in Wild Birds.

Injuries and bone fractures are the most frequent causes of admission at wildlife rescue centers. Wild birds are more susceptible to open fractures due to their anatomical structure, which can lead to osteomyelitis and necrosis. Antibiotic therapy in these cases is indispensable, but the increase of antimicrobial-resistant isolates in wildlife has become a significant concern in recent years. In this context, the likelihood of antibiotic failure and death of animals with infectious issues is high. This study aimed to isolate, identify, and assess the antimicrobial resistance pattern of bacteria in wounds and open fractures in wild birds. To this end, injured birds admitted to a wildlife rescue center were sampled, and bacterial isolation and identification were performed. Then, antimicrobial susceptibility testing was assessed according to the disk diffusion method. In total, 36 isolates were obtained from 26 different birds. The genera detected were Staphylococcus spp. (63.8%), Escherichia (13.9%), Bacillus (11.1%), Streptococcus (8.3%), and Micrococcus (2.8%). Among Staphylococcus isolates, S. lentus and S. aureus were the most frequent species. Antimicrobial resistance was detected in 82.6% of the isolates, among which clindamycin resistance stood out, and 31.6% of resistant isolates were considered multidrug-resistant. Results from this study highlight the escalating scope of antimicrobial resistance in wildlife. This level of resistance poses a dual concern for wildlife: firstly, the risk of therapeutic failure in species of significant environmental value, and, secondly, the circulation of resistant bacteria in ecosystems.

In vitro selection predicts malaria parasite resistance to dihydroorotate dehydrogenase inhibitors in a mouse infection model.

Resistance has developed in Plasmodium malaria parasites to every antimalarial drug in clinical use, prompting the need to characterize the pathways mediating resistance. Here, we report a framework for assessing development of resistance of Plasmodium falciparum to new antimalarial therapeutics. We investigated development of resistance by P. falciparum to the dihydroorotate dehydrogenase (DHODH) inhibitors DSM265 and DSM267 in tissue culture and in a mouse model of P. falciparum infection. We found that resistance to these drugs arose rapidly both in vitro and in vivo. We identified 13 point mutations mediating resistance in the parasite DHODH in vitro that overlapped with the DHODH mutations that arose in the mouse infection model. Mutations in DHODH conferred increased resistance (ranging from 2- to ~400-fold) to DHODH inhibitors in P. falciparum in vitro and in vivo. We further demonstrated that the drug-resistant parasites carrying the C276Y mutation had mitochondrial energetics comparable to the wild-type parasite and also retained their fitness in competitive growth experiments. Our data suggest that in vitro selection of drug-resistant P. falciparum can predict development of resistance in a mouse model of malaria infection.

Egg Yolk Provides Th2 Adjuvant Stimuli and Promotes Sensitization to Egg White Allergens in BALB/c Mice.

SCOPE: Egg is the second most frequent source of allergic reactions in children. Egg yolk (EY) amounts to one-third in weight of a fresh whole egg, but its contribution to egg allergy has not been investigated in depth. This study assesses whether EY influences the capacity of egg white (EW) to sensitize and trigger allergic responses. METHODS AND RESULTS: BALB/c mice were exposed to EW, EY, and their mixture, using models of orally (with and without adjuvant) and adjuvant-free intraperitoneally induced allergy. In vitro assays were also conducted to examine epithelial and dendritic cell (DC) functions. Results showed that EY played a role during the sensitizing phase of allergy. EY exerted local Th2-biasing effects through the upregulation of intestinal IL-33 expression and it also favored Th2 polarization directly during DC presentation of allergens to T cells. CONCLUSION: The results obtained reveal that EY provides Th2-adjuvant stimuli to the immune system that may increase the susceptibility to develop egg allergy. The joint administration of EW and EY may be a trigger for initiation or maintenance of egg allergy with implications in prevention strategies regarding egg introduction in the diet of susceptible children.

Seroprevalence of Toxoplasma gondii in Iberian pig sows.

The objective of the present study was to investigate the seroprevalence of Toxoplasma gondii infection in Iberian sows reared in extensive and intensive management conditions and to compare two serologic techniques used for diagnosis. In addition, some possible risk factors associated with the presence of serum antibodies to T. gondii were also studied. Serum samples were collected from 2492 Iberian sows on 14 pig farms. Three types of management systems were included, traditional extensive outdoor farms (five farms), intensive farms with outdoor access (n = 4), and conventional intensive indoor farms (n = 5). The presence of serum antibodies to T. gondii was evaluated by two commercially available tests: an indirect enzyme-linked immunosorbent assay (ELISA) and a direct agglutination test (DAT). Serum antibodies against T. gondii were detected in 237 sows (9.5%) by at least one of the techniques used. The mean seroprevalence of toxoplasmosis in Iberian sows was 5.8% by ELISA and 8.9% by DAT. An agreement kappa-value of 0.68 (95%, CI = 0.63-0.74) was found between both tests. The results from this study suggest that the prevalence of T. gondii antibodies among Iberian sows seems to be moderate-low. The presence of serum antibodies against T. gondii in Iberian sows was associated to an extensive management system and low-level facilities of the farm, sow number (> 1000 animals), presence of cats, absence of rodent control and bird-proof nets in windows, well-water source, feed sources and storage (from the same farm and not stored in silo), absence of fences, and low farm worker qualification.

Assessment of the Allergenic Potential of the Main Egg White Proteins in BALB/c Mice.

This work aimed to assess the contribution of the major egg white proteins, ovalbumin, ovomucoid, and lysozyme, to the induction and elicitation of allergenic responses. For this purpose, BALB/c mice were orally administered either the individual egg allergens or a mixture of the three proteins in the same proportion, to evaluate their relative allergenicity avoiding their different abundance in egg white. Cholera toxin was used as a T helper 2 (Th2)-polarizing adjuvant. Ovomucoid and lysozyme triggered the most severe anaphylaxis reactions upon oral challenge. In comparison to ovalbumin and ovomucoid, lysozyme was a more active promotor of early immunoglobulin E and immunoglobulin G1 production and stimulated stronger Th2-biased responses from both mesenteric lymph node and spleen cells. These results indicate that lysozyme is highly immunogenic and should be considered as a major allergen, whose clinical usefulness in the diagnosis, prognosis, and therapeutic approaches of egg allergy deserves further consideration.

Dietary Intervention with ß-Lactoglobulin-Derived Peptides and a Specific Mixture of Fructo-Oligosaccharides and Bifidobacterium breve M-16V Facilitates the Prevention of Whey-Induced Allergy in Mice by Supporting a Tolerance-Prone Immune Environment.

Cow's milk allergy (CMA) prevails in infants and brings increased risk of developing other allergic diseases. Oral administration of specific ß-lactoglobulin (BLG)-derived peptides (PepMix) and a specific blend of short- and long-chain fructo-oligosaccharides and Bifidobacterium breve M-16V (FF/Bb) was found to partially prevent CMA development in mice. In this study, we aimed to expand the knowledge on the preventive potential and the underlying mechanisms of this approach. Three-week-old female C3H/HeOuJ mice were orally exposed to PepMix±FF/Bb prior to a 5-week oral sensitization with whole whey and cholera toxin as an adjuvant. The acute allergic skin response was determined after an intradermal challenge with whole whey protein. Following an oral challenge with whey, regulatory T cells (Tregs) in the small intestine lamina propria (SI-LP) and mRNA expression of immune markers in the Peyer's patches (PP) were investigated. The early impact of PepMix and FF/Bb interventions on the immune system during the oral tolerance (OT) induction phase was investigated after the last OT administration. Pre-exposing mice to PepMix+FF/Bb partially prevented the acute allergic skin response compared to PBS and increased Tregs and activated T cells in the SI-LP compared to sham-sensitized mice. It also increased the mRNA expression of Tbet over GATA3 in the PP of whey-sensitized mice. Directly upon the 6-day OT phase, FF/Bb intervention enhanced cecal content levels of propionic and butyric acid in PepMix-fed mice and the former was positively correlated with Foxp3+ cell numbers in the colon. In the PP of PepMix+FF/Bb-exposed mice, IL-22 mRNA expression increased and IL-10 followed the same tendency, while the Foxp3 expression was increased over GATA3 and RorγT. In the colon, the Tbet mRNA expression increased over GATA3, while IL-22 decreased. In addition, the Foxp3+/GATA3+ and regulatory/effector T cell ratios in the mesenteric lymph nodes and the CD11b+/CD11b- conventional dendritic cells ratio in the SI-LP were increased. In conclusion, the FF/Bb diet facilitates the capacity of the specific BLG-peptides to partially prevent the allergic response after sensitization to whole whey protein, possibly by creating a tolerance-prone environment during the OT phase. Such a dietary intervention might contribute to tailoring successful strategies for CMA prevention.

Sensitizing and Eliciting Capacity of Egg White Proteins in BALB/c Mice As Affected by Processing.

This study assesses to what extent technological processes that lead to different degrees of denaturation of egg white proteins affect their allergenicity. We focused on heat (80 °C, 10 min) and high-pressure (400 MPa and 37 °C, 10 min) treatments and used a BALB/c mouse model of food allergy. Oral sensitization to egg white using cholera toxin as adjuvant induced the production of IgE and IgG1 isotypes and led to severe clinical signs following challenge with the allergen. Extensive protein denaturation caused by heat treatment increased its ability to induce Th1 responses and reduced both its sensitizing and eliciting capacity. Heated egg white stimulated the production of IgE over IgG1 antibodies directed, at least in part, toward new epitopes exposed as a result of heat treatment. Conversely, partial denaturation caused by high-pressure treatment increased the ability of egg white to stimulate Th2 responses and its allergenic potential.

Antibody Production, Anaphylactic Signs, and T-Cell Responses Induced by Oral Sensitization With Ovalbumin in BALB/c and C3H/HeOuJ Mice.

PURPOSE: Two mouse strains, BALB/c and C3H/HeOuJ, broadly used in the field of food allergy, were compared for the evaluation of the allergenic potential of ovalbumin (OVA). METHODS: Sensitization was made by administering 2 different OVA doses (1 and 5 mg), with cholera toxin as Th2-polarizing adjuvant. Antibody levels, severity of anaphylaxis, and Th1 and Th2 responses induced by the allergen were assessed. In addition, because the mice selected had functional toll-like receptor 4, the influence of contamination with lipopolysaccharide (LPS) on the immunostimulating capacity of OVA on spleen cells was also evaluated. RESULTS: Both strains exhibited similar susceptibility to OVA sensitization. The 2 protein doses generated similar OVA-specific IgE and IgG1 levels in both strains, whereas C3H/HeOuJ mice produced significantly more IgG2a. Oral challenge provoked more severe manifestations in C3H/HeOuJ mice as indicated by the drop in body temperature and the severity of the anaphylactic scores. Stimulation of splenocytes with OVA led to significantly higher levels of Th2 and Th1 cytokines in BALB/c, and these were less affected by protein contamination with LPS. CONCLUSIONS: The antibody and cytokine levels induced by OVA in BALB/c mice and the observation that BALB/c spleen cell cultures were more resistant than those of C3H/HeOuJ mice to the stimulus of LPS make this strain prone to exhibit Th2-mediated food allergic reactions and very adequate for the study of the features of OVA that make it allergenic.

Presence of Ostertagia ostertagi antibodies in bulk tank milk from cattle herds in northern Spain.

The objective of this survey was to investigate the Ostertagia ostertagi infection status of cattle dairy herds from northern Spain through measurement of antibody concentration in the bulk tank milk (BTM). In addition, management and performance data were collected to determine possible relationships with BTM O. ostertagi antibody levels. BTM samples were collected in 118 dairy cattle farms from five northern Spain Autonomic Communities in the autumn of 2009 and spring of 2010. The O. ostertagi antibody levels in milk were determined using a commercial ELISA kit (Svanovir) and farm management information included type and farm size, production level, access to pasture, grazing regime and anthelmintic treatments. The overall mean optical density ratio (ODR) values of the two sampling times ranged from 0.60 (0.08-1.32) in autumn to 0.56 (0.05-1.24) in spring and no seasonal differences were detected. Herds with access to pasture showed significantly higher titers of O. ostertagi antibodies in milk (mean ODR=0.69-0.63) than in those without access (mean ODR=0.36-0.33). The association analyses showed significant correlations between the BTM O. ostertagi ODR and location (Navarra showed the highest ODR values), herd size (ODR decreased with increased herd size), milk production level (ODR negatively associated with milk yield), calves access to pasture (higher ODR when calves <12 months had access to pasture), outside access (no access low ODR, access high ODR) and grazing management (ODR increased proportionally to the percentage of time spent grazing each day). This study emphasizes that O. ostertagi-induced production losses should be considered on dairy farms in northern Spain. Additionally, putative risk-factors should be also considered for preventive measures.