Effect of honey bee forage plants in Tunisia on diversity and antibacterial potential of lactic acid bacteria and bifidobacteria from Apis mellifera intermissa and its products.

Lactic acid bacteria and bifidobacteria (LAB and Bifido), isolated from the gastrointestinal tract of Apis mellifera intermissa (BGIT), honey (H), propolis (P) and bee bread (BB) of hives set in different vegetations (wildflowers, caraway, orange blossom, Marrubium vulgare, Eucalyptus and Erica cinerea), were subjected to analysis of their antibacterial potential. Isolates able to inhibit Staphylococcus aureus were selected and identified with MALDI-TOF MS leading to 154 strains representing 12 LAB and Bifido species. Lactiplantibacillus plantarum, Pediococcus pentosaceus and Enterococcus faecalis were predominantly found in all matrices. BGIT showed the highest LAB and Bifido diversity with exclusive occurrences of five species (including Bifidobacterium asteroides and Limosilactobacillus fermentum). Honey was the second origin harboring an important variety of LAB species of which Apilactobacillus kunkeei and Enterococcus mundtii were characteristic of both H and BGIT. Principal components analysis revealed associations between antibacterial activities of LAB and Bifido, matrices and honey bee forage plants. Inhibition trends of S. aureus and Citrobacter freundii were highlighted with: L. plantarum from BGIT, P, H of bees feeding on E. cinerea; Pediococcus pentosaceus from BGIT, P, BB associated with E. cinerea; and Bifidobacterium asteroides from BGIT/orange blossom system. However, Enterococcus faecium associated with BGIT/Eucalyptus system antagonized Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. Our findings highlighted noteworthy effects of bee forage plants on the antibacterial activity of LAB and Bifido. Our approach could be useful to identify multiple conditions promoting antibacterial potency of LAB and Bifido under the combined effects of feeding plants and living matrices.

Bilateral mastocytic and fibroblastic nodular proliferative dermatosis affecting ear margins in three domestic cats.

This case series describes the clinical appearance, histopathological findings and therapeutic trials of proliferative nodular lesions on bilateral ear margins of three domestic cats including two littermates. All therapeutic trials were unsuccessful. While the aetiology remains unclear, this report highlights different hypotheses in presenting this unusual inflammatory and fibroblastic dermatosis in cats.

Alpha-amylase and alphaglucosidase inhibitory properties, beta-galactosidase activity, and probiotic potential of lactic acid bacteria and bifidobacteria from Apis mellifera intermissa and its products.

The present study aimed to evaluate the probiotic potential, α-amylase and α-glucosidase inhibitory effects, and ß-galactosidase production of 19 non haemolytic lactic acid bacteria and bifidobacteria previously identified and isolated from honey bee gastrointestinal tract (BGIT) of Apis mellifera intermissa, honey, propolis and bee bread. The isolates were screened according to their high resistance to lysozyme and potent antibacterial activity. Our results indicated that among the 19 isolates, Limosilactobacillus fermentum BGITE12.2, Lactiplantibacillus plantarum BGITEC13, Limosilactobacillus fermentum BGITEC5.1 and Bifidobacterium asteroides BGITOB8, isolated from BGIT exhibited a good tolerance to 100 mg/mL lysozyme (> 82%), excellent tolerance to 0.5% bile salt [survival rate (SR) ≥ 83.19% ± 0.01], and a high SR (≥ 80.0%) under gastrointestinal tract conditions. The auto-aggregation ability was high (auto-aggregation index ranging from 67.14 ± 0.16 to 92.8% ± 0.03) for L. fermentum BGITE12.2, L. plantarum BGITEC13, and B. asteroides BGITOB8, and moderate for L. fermentum BGITEC5.1 (39.08% ± 0.11). Overall, the four isolates showed moderate co-aggregation capacity with pathogenic bacteria. They exhibited from moderate to high hydrophobicity towards toluene and xylene. The safety assessment revealed that the four isolates lacked gelatinase and mucinolytic activities. Also, they were susceptible to ampicillin, clindamycin, erythromycin, and chloramphenicol. Interestingly, the four isolates showed α-glucosidase and α-amylase inhibitory activities ranging from 37.08 ± 0.12 to 57.57% ± 0.1 and from 68.30 ± 0.09 to 79.42% ± 0.09, respectively. Moreover, L. fermentum BGITE12.2, L. plantarum BGITEC13, L. fermentum BGITEC5.1 isolates exhibited ß-galactosidase activity over a wide range of 52.49 ± 0.24-746.54 ± 0.25 Miller Units. In conclusion, our findings suggest that the four isolates could be potential candidates for probiotics with interesting functional properties.

Inflammatory Monocytes and Neutrophils Regulate Streptococcus suis-Induced Systemic Inflammation and Disease but Are Not Critical for the Development of Central Nervous System Disease in a Mouse Model of Infection.

Streptococcus suis is an important porcine bacterial pathogen and zoonotic agent responsible for sudden death, septic shock, and meningitis. These pathologies are a consequence of elevated bacterial replication leading to exacerbated and uncontrolled inflammation, a hallmark of the S. suis systemic and central nervous system (CNS) infections. Monocytes and neutrophils are immune cells involved in various functions, including proinflammatory mediator production. Moreover, monocytes are composed of two main subsets: shorter-lived inflammatory monocytes and longer-lived patrolling monocytes. However, regardless of their presence in blood and the fact that S. suis-induced meningitis is characterized by infiltration of monocytes and neutrophils into the CNS, their role during the S. suis systemic and CNS diseases remains unknown. Consequently, we hypothesized that monocytes and neutrophils participate in S. suis infection via bacterial clearance and inflammation. Results demonstrated that inflammatory monocytes and neutrophils regulate S. suis-induced systemic disease via their role in inflammation required for bacterial burden control. In the CNS, inflammatory monocytes contributed to exacerbation of S. suis-induced local inflammation, while neutrophils participated in bacterial burden control. However, development of clinical CNS disease was independent of both cell types, indicating that resident immune cells are mostly responsible for S. suis-induced CNS inflammation and clinical disease and that inflammatory monocyte and neutrophil infiltration is a consequence of the induced inflammation. In contrast, the implication of patrolling monocytes was minimal throughout the S. suis infection. Consequently, this study demonstrates that while inflammatory monocytes and neutrophils modulate S. suis-induced systemic inflammation and disease, they are not critical for CNS disease development.

Differential role of MyD88 signaling in Streptococcus suis serotype 2-induced systemic and central nervous system diseases.

Streptococcus suis serotype 2 is an important porcine bacterial pathogen and a zoonotic agent responsible for sudden death, septic shock and meningitis, with exacerbated inflammation being a hallmark of the systemic and central nervous system (CNS) infections. However, S. suis serotype 2 strains are genetically and phenotypically heterogeneous, being composed of a multitude of sequence types (STs) whose virulence greatly varies. Yet, most studies have used 'classical' virulent Eurasian ST1 or ST7 strains, even though ST25 and ST28 strains account for most isolates in North America. While recognition of S. suis by innate immune cells has been associated with the myeloid differentiation primary response 88 (MyD88)-dependent Toll-like receptor (TLR) pathway in vitro, particularly surface-associated TLR2, little information is available regarding its role in vivo. This study demonstrates for the first time a differential role of MyD88 signaling in S. suis-induced systemic and CNS diseases, regardless of strain background diversity. The MyD88-dependent pathway is critical for the development of systemic disease via its role in inflammation, which subsequently controls bacterial burden. However, and differently from what has been described in vitro, TLR2 and TLR4 individually do not contribute to systemic disease, suggesting possible compensation in their absence and/or a collaborative role with other MyD88-dependent TLRs. On the other hand, CNS disease does not necessarily require MyD88 signaling and, consequently, neither TLR2 nor TLR4, suggesting a partial implication of other pathways. Finally, regardless of its notable heterogeneity, recognition of S. suis serotype 2 appears to be similar, indicating that recognized components are conserved motifs.

Neonatal and Juvenile Ocular Development in Göttingen Minipigs and Domestic Pigs: A Histomorphological and Immunohistochemical Study.

Pigs are considered one of the relevant animal models for ocular research as they share several histological and anatomical similarities with the human eye. With the increasing interest in juvenile animal models, this study aimed to describe the postnatal development of ocular structures in 16 Göttingen minipigs and 25 F2 domestic pigs, between birth and 6 months of age, using histopathology and immunohistochemistry against Ki-67, caspase-3, calbindin, glial fibrillary acidic protein, rhodopsin, and synaptophysin. All ocular structures in both pig breeds were incompletely developed at birth and for variable periods postnatally. Noteworthy histological features of immaturity included vascularization in the corneal stroma in neonatal Göttingen minipigs, increased cellularity in different substructures, remnants of the hyaloid vasculature, short and poorly ramified ciliary body processes, and a poorly developed cone inner segment. Increased cellular proliferation, highlighted by abundant Ki-67 immunolabeling, was observed in almost all developing structures of the pig eye for variable periods postnatally. Apoptosis, highlighted with caspase-3 immunolabeling, was observed in the retinal inner nuclear layer at birth and in the regressing hyaloid vasculature remnants. Immunohistochemistry against rhodopsin, synaptophysin, and calbindin demonstrated the short size of the developing photoreceptors and the immature cone inner segment morphology. Calbindin labeling revealed significant differences in the amount of positively labeled cone nuclei between the retinal area centralis and the non-area centralis regions. The elongation of Müller cell processes in the developing retina was shown with glial fibrillary acidic protein. In both pig breeds, the eyes reached histomorphological and immunohistochemical maturity at 6 months of age.

Histomorphologic Analysis of the Late-term Rat Fetus and Placenta.

Histological examination of the rat placenta and fetus is uncommon. Toxicological studies mainly rely on gross examination of the fetus and on fetal and placental weights. These are often insufficient to assess the fetal and placental toxicity of xenobiotics. The small size of the fetus makes its dissection labor-intensive. Thus, our objective was to develop a simple and accurate technique to evaluate the rat fetus and placenta. Sprague-Dawley rat fetuses at gestational day 19.5 ( n = 18) and their placentas ( n = 32) were fixed in formalin. Placentas were cut transversally in the center. Fetuses were cut following a freehand whole-body serial sectioning diagram adapted from Wilson's method. Sections were stained with hematoxylin-eosin-phloxine-saffron, and histomorphometry was used to measure the area of the fetal placental region (27.2 ± 1.7 mm2), including the labyrinth (22.2 ± 1.0 mm2) and the basal zone (4.8 ± 0.8 mm2). Our whole-fetus serial sectioning technique resulted in 12 precise cutting planes that fit on 3 histological slides, enabling the examination of most organs without labor-intensive dissection. Quantitative analysis of placental areas improves the understanding of the pathogenesis of treatment-related changes. This technique provides a standardized method for future research in pertinent fields such as developmental biology and toxicology.

Neonatal and Juvenile Ocular Development in Sprague-Dawley Rats: A Histomorphological and Immunohistochemical Study.

As in many altricial species, rats are born with fused eyelids and markedly underdeveloped eyes. While the normal histology of the eyes of mature rats is known, the histomorphological changes occurring during postnatal eye development in this species remain incompletely characterized. This study was conducted to describe the postnatal development of ocular structures in Sprague-Dawley (SD) rats during the first month of age using histology and immunohistochemistry (IHC). Both eyes were collected from 51 SD rats at 13 time points between postnatal day (PND)1 and PND30. Histologic examination of hematoxylin and eosin-stained sections was performed, as well as IHC for cleaved-caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to evaluate apoptosis, and IHC for Ki-67 and phospho-histone-H3 to evaluate cell proliferation. Extensive ocular tissue remodeling occurred prior to the eyelid opening around PND14 and reflected the interplay between apoptosis and cell proliferation. Apoptosis was particularly remarkable in the maturing subcapsular anterior epithelium of the lens, the inner nuclear and ganglion cell layers of the developing retina, and the Harderian gland, and was involved in the regression of the hyaloid vasculature. Nuclear degradation in the newly formed secondary lens fibers was noteworthy after birth and was associated with TUNEL-positive nuclear remnants lining the lens organelle-free zone. Cell proliferation was marked in the developing retina, cornea, iris, ciliary body and Harderian gland. The rat eye reached histomorphological maturity at PND21 after a rapid phase of morphological changes characterized by the coexistence of cell death and proliferation.

Hemangiosarcoma within an intermuscular lipoma in a golden retriever dog.

A subcutaneous mass on the right pelvic limb of an 11-year-old neutered male golden retriever dog was surgically excised. A hemangiosarcoma included within an intermuscular lipoma was diagnosed upon histopathological examination. To the authors' knowledge, this is the first case report of this nature in a dog.

Hémangiosarcome dans un lipome intermusculaire chez un golden retriever. Une masse sous-cutanée située sur le membre pelvien droit d'un golden retriever mâle castré de 11 ans a été excisée chirurgicalement. Un hémangiosarcome inclus dans un lipome intermusculaire a été diagnostiqué à l'examen histopathologique. Selon les auteurs, il s'agirait du premier rapport de cas de ce type chez un chien.(Traduit par les auteurs).

A Rare UGT2B7 Variant Creates a Novel N-Glycosylation Site at Codon 121 with Impaired Enzyme Activity.

The UDP glucuronosyltransferase (UGT) superfamily comprises glycoproteins that reside in the endoplasmic reticulum membranes and that undergo post-translational modifications (PTMs). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and examine only a small fraction of the genetic diversity; however, rare variants (frequency <1%) might have a significant effect because they are predicted to greatly outnumber common variants in the human genome. We discovered a rare single nucleotide UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino acid substitution at codon 121. This low-frequency variation, found in two individuals of a population of 305 healthy volunteers, leads to the translation of an asparagine instead of an aspartic acid (UGT2B7 p.D121N). This amino acid change was predicted to create a putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced UGT2B7 with tunicamycin in human embryonic kidney (HEK293) cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease of 49% and 40% in the formation of zidovudine and mycophenolic acid glucuronides, respectively. A systematic survey of the Short Genetic Variations database uncovered 32 rare, naturally occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in the other UGT2B enzymes. Collectively, these variants have the potential to increase the proportion of variance explained in the UGT pathway resulting from changes in PTMs, such as N-linked glycosylation with consequences on drug metabolism.

Inhibition of islet amyloid polypeptide aggregation and associated cytotoxicity by nonsteroidal anti-inflammatory drugs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) constitute an important pharmacotherapeutic class that, over the past decade, have expanded in application to a panoply of medical conditions. They have been tested for neurodegenerative diseases such as Alzheimer's to reduce inflammation and also in the attempt to abrogate amyloid deposition. However, the use of NSAIDs as aggregation inhibitors has not been extensively studied in pancreatic amyloid deposition. Pancreatic amyloidosis involves the misfolding of islet amyloid polypeptide (IAPP) and contributes to the progression of type-2 diabetes in humans and felines. To ascertain their antiamyloidogenic activity, several NSAIDs were tested using fluorometric thioflavin-T assays, circular dichroism, photo-induced cross-linking assays, and cell culture. Celecoxib, diclofenac, indomethacin, meloxicam, niflumic acid, nimesulide, phenylbutazone, piroxicam, sulindac, and tenoxicam reduced fibrillization at a molar ratio of 1:10. The circular dichroism spectra of diclofenac, piroxicam, and sulindac showed characteristic spectral signatures found in predominantly α-helical structures. The oligomerization of human IAPP was abrogated with diclofenac and sulindac at a molar ratio of 1:5. The cytotoxic effects of pre-incubated human IAPP on cultured INS-1 cells were noticeably reduced in the presence of diclofenac, meloxicam, phenylbutazone, sulindac, and tenoxicam at a molar ratio of 1:10. Our results demonstrate that NSAIDs can provide chemical scaffolds to generate new and promising antiamyloidogenic agents that can be used alone or as a coadjuvant therapy.

Discovery of ethyl urea derivatives as inhibitors of islet amyloid polypeptide fibrillization and cytotoxicity.

Islet amyloid polypeptide (IAPP) has been shown to form amyloid deposits in pancreatic islets, thereby furthering type 2 diabetes disease progression. Further discovery of new molecules is needed to create a diverse set of molecules that impede pancreatic amyloidosis. We have recently designed and synthesized N-phenyl-N'-(2-ethyl)ureas (EU) that are non-cytotoxic small molecules, to evaluate the role of the aryl-substituted moiety on the inhibition of hIAPP fibrillization. Several EUs were tested in vitro for their anti-amyloidogenic activity using the fluorometric ThT assay, the photo-induced cross-linking (PIUCP) assay, and cell survival assay in pancreatic MIN-6 cells. EU-362 and EU-418 were able to significantly inhibit the formation of hIAPP fibrils and protected cells from amyloid cytotoxic effects. Our results suggest that increasing the nucleophilic potency of the aryl moiety significantly enhances the anti-amyloidogenic activity of the molecules.

Identification of 5α-reductase isoenzymes in canine skin.

BACKGROUND: Alopecia X in dogs is a noninflammatory alopecia that may be caused by a hormonal dysfunction. It may be similar to androgenic alopecia in men that is caused by the effect of dihydrotestosterone (DHT). The 5α-reductase isoenzymes, 5αR1 and 5αR2, and a recently described 5αR3, are responsible for the conversion of testosterone into DHT. However, which 5α-reductases are present in canine skin has not yet been described. OBJECTIVES: The main objective of this study was to determine the pattern of expression of 5α-reductase genes in canine skin. METHODS: Skin biopsies were obtained from healthy, intact young-mature beagles (three males, four females) at three anatomical sites normally affected by alopecia X (dorsal neck, back of thighs and base of tail) and two sites generally unaffected (dorsal head and ventral thorax). Prostate samples (n = 3) were collected as positive controls for 5α-reductase mRNA abundance measurement by real-time PCR. RESULTS: We detected mRNA encoding 5αR1 and 5αR3 but not 5αR2. There were no significant differences in 5αR1 and 5αR3 mRNA levels between the different anatomical sites, irrespective of gender (P > 0.05). Moreover, the mean mRNA abundance in each anatomical site did not differ between males and females (P > 0.05). CONCLUSIONS AND CLINICAL IMPORTANCE: To the best of the authors' knowledge, this is the first study demonstrating the expression of 5α-reductases in canine skin and the expression of 5αR3 in this tissue. These results may help to elucidate the pathogenesis of alopecia X and to determine more appropriate treatments for this disorder.

Cutaneous squamous cell carcinoma in an African pygmy hedgehog (Atelerix albiventris).

A cutaneous mass was surgically excised in a 4-year-old African pygmy hedgehog (Atelerix albiventris). A squamous cell carcinoma was diagnosed based on histopathological examination and local recurrence following excision is strongly suspected. To the authors' knowledge, this is the first well-documented report of a cutaneous squamous cell carcinoma in this species.

Carcinome squameux cutané chez un hérisson pygmée d'Afrique(Atelerix albiventris). Une masse cutanée a été excisée par chirurgie chez un hérisson pygmée d'Afrique âgé de 4 ans (Atelerix albiventris). Un carcinome squameux a été diagnostiqué en se basant sur un examen histopathologique et la récurrence locale est fortement soupçonnée après l'excision. À la connaissance des auteurs, il s'agit du premier rapport bien documenté d'un carcinome squameux chez cette espèce.(Traduit par Isabelle Vallières).

Characterization of a pancreatic islet cell tumor in a polar bear (Ursus maritimus).

Herein, we report a 25-year-old male polar bear suffering from a pancreatic islet cell tumor. The aim of this report is to present a case of this rare tumor in a captive polar bear. The implication of potential risk factors such as high carbohydrate diet or the presence of amyloid fibril deposits was assessed. Necropsy examination revealed several other changes, including nodules observed in the liver, spleen, pancreas, intestine, and thyroid glands that were submitted for histopathologic analysis. Interestingly, the multiple neoplastic nodules were unrelated and included a pancreatic islet cell tumor. Immunohistochemistry of the pancreas confirmed the presence of insulin and islet amyloid polypeptide (IAPP) within the pancreatic islet cells. The IAPP gene was extracted from the paraffin-embedded liver tissue and sequenced. IAPP cDNA from the polar bear exhibits some differences as compared to the sequence published for several other species. Different factors responsible for neoplasms in bears such as diet, infectious agents, and industrial chemical exposure are reviewed. This case report raised several issues that further studies may address by evaluating the prevalence of cancers in captive or wild animals.

Optical Coherence Tomography: A Review of Current Applications in Veterinary Ophthalmology.

Normative standards for healthy animal structures have been established by optical coherence tomography (OCT). OCT has been used in animal studies to characterize more precisely ocular lesions, identify the origin of the affected layer, and eventually provide a curative treatment. To acquire a high image resolution, several challenges must be overcome when performing an OCT scan on animals. Sedation or general anesthesia is usually necessary in OCT image acquisition to alleviate motion during image acquisition. Mydriasis, eye position and movements, head position, and corneal hydration must also be managed during the OCT analysis.

Histology of the whole body of honey bees: tissue fixation and processing.

Beekeeping plays a crucial role in biodiversity, pollination, commercial farming, and the worldwide agricultural economy. Histopathology, which is an important tool for the investigation of diseases in vertebrates, is not commonly used in honey bees (Apis mellifera). However, histopathology could potentially help the diagnostic investigation of high mortality in bees. We developed a tissue fixation and processing method enabling systematic production of histologic slides adequate for diagnostic and research purposes. Our method uses inexpensive, accessible products and can be realized with conventional pathology laboratory equipment. The quality of histologic slides obtained is similar to those of vertebrate animals processed routinely in pathology laboratories. Histopathology as a diagnostic and research tool will improve the services currently offered to apiarists and could help decrease the mean mortality rate, increase apiarists' profits, and ensure long-term pollination services.

Morphological Evaluation of Transscleral Laser Retinopexy in Rabbits: Comparison of Optical Coherence Tomography and Histologic Examinations.

Transscleral retinopexy is a preventive technique used against retinal detachment. Fundus examination can allow the monitoring of morphological retinal changes in the progression of photocoagulation lesions, without offering details on the morphological changes by the retinal lesion. The aim of the study was to assess the progression of photocoagulation lesions induced by transscleral retinopexy (840 nm diode laser), by comparing the optical coherence tomography (OCT) and histological images over a period of six weeks on eight pigmented New Zealand healthy rabbits (four males and four females; n = 16 eyes). All rabbits underwent transscleral retinopexy on their left eye on day 0 (D0). Measurements of the photocoagulation lesions were obtained in vivo on D0, D7, D15, D21, and D42 by acquiring OCT images of both eyes from all rabbits. On D1, D7, D21, and D42, two rabbits were euthanized, and their eyes were enucleated. A significant effect by time on the decrease in the central retinal thickness of the photocoagulation lesion was observed from D1 to D7 (p = 0.001); however, no such effect was observed on the horizontal length ((HL) p = 0.584) of the lesion surface. The reliability between the OCT and histological measurements, which were evaluated using intraclass correlation coefficients, was excellent for measuring the retinal thickness at the center (ICC = 0.91, p < 0.001), moderate for the right side of the retinal lesions (ICC = 0.72, p = 0.006), and not significant for the left side and HL (p = 0.055 and 0.500, respectively). The morphological changes observed in the OCT and histopathological images of the photocoagulation lesions were qualitatively described over time. OCT is an effective tool for monitoring changes in photocoagulation lesions. Some measurements and qualitative changes showed an adequate correlation between the OCT and histological findings.

Establishment of preliminary reference intervals and cytochemical staining of blood cells in big-bellied seahorses (Hippocampus abdominalis).

BACKGROUND: Despite their popularity, hematology reference intervals (RIs) have not been established in big-bellied seahorses (Hippocampus abdominalis). OBJECTIVE: The objectives of this study were to establish hematologic RIs to compare values between sex in regard to cytochemical staining of blood cells. We also sought to compare white blood cell concentrations using the Natt and Herrick technique vs blood smear estimates. METHODS: Forty-three healthy individuals from the Aquarium du Québec (22 females and 21 males) were included. Normal health status was confirmed by an unremarkable physical examination in five individuals and by necropsy of five other individuals, of which all were excluded from further analyses. Venipuncture was performed from the ventral coccygeal vein in the remaining 33 individuals without anesthesia using heparinized insulin syringes. A blood volume of 0.05 to 0.1 ml was collected to prepare Wright Giemsa-stained blood smears and hematocrits immediately after venipuncture. Whole blood was stored in heparinized Eppendorf tubes to determine red and white blood cell concentrations using the Natt and Herrick technique with a hemocytometer in 10 individuals; these results were compared with blood smear estimates. Additional blood smears were stained with alkaline phosphatase substrate, periodic acid Schiff, and toluidine blue stains. RESULTS: The reference intervals included the packed cell volume (27.4-67.5%), thrombocyte count (19.5-197.7 × 109 /L), and white blood cell (WBC) count (2-54.8 × 109 /L), including neutrophils (1.1-21.3 × 109 /L), lymphocytes (2.7-45.5 × 109 /L), and monocytes (0-2.2 × 109 /L). The WBC hemocytometer counts showed no correlation with blood smear estimates (Spearman's rho = 0.2). There was also no significant difference between the sexes. CONCLUSIONS: These preliminary reference intervals will help assess the health of seahorses.

Nanoparticle-Enabled Combination Therapy Showed Superior Activity against Multi-Drug Resistant Bacterial Pathogens in Comparison to Free Drugs.

The emergence of multidrug-resistant (MDR) bacterial pathogens in farm animals and their zoonotic spread is a concern to both animal agriculture and public health. Apart from antimicrobial resistance (AMR), bacterial pathogens from the genera of Salmonella and Staphylococcus take refuge inside host cells, thereby demanding intervention strategies that can eliminate intracellular MDR pathogens. In this study, seven clinical isolates of Salmonella and Staphylococcus from swine farms were characterized for antibiotic (n = 24) resistance, resistance mechanisms, and virulence characteristics. All isolates showed resistance to one or more antibiotics and S. enterica ser. Typhimurium isolate had the highest resistance to the panel of antibiotics tested. Major resistance mechanisms identified were efflux pump and beta-lactamase enzyme activities. Staphylococcus isolates showed complete hemolysis and strong biofilm formation, while Salmonella isolates caused partial hemolysis, but showed no or weak biofilm formation. MDR isolates of S. aureus M12 and S. enterica ser. Typhimurium bacteria were subsequently tested against combinations of antibiotics and potentiating adjuvants for improved antibacterial efficacy using a checkerboard assay, and their fractional inhibitory concentration index (FICI) was calculated. A combination of chitosan and silica nanoparticles containing tetracycline (TET) and efflux pump inhibitor chlorpromazine (CPZ), respectively, was characterized for physicochemical properties and effectiveness against MDR Salmonella enterica ser. Typhimurium isolate. This combination of nano-encapsulated drugs improved the antibacterial efficacy by inhibiting AMR mechanisms (efflux activity, beta-lactamase enzyme activity, and hydrogen sulfide (H2S) production) and reducing intracellular pathogen load by 83.02 ± 14.35%. In conclusion, this study sheds light on the promising applicability of nanoparticle-enabled combination therapy to combat multidrug-resistant pathogens encountered in animal agriculture.