Using fluorescently labeled African Swine Fever Virus (ASFV) strains, a high (~0.2) quasi-recombinant frequency (qRF3-4) was detected ex vivo during 3–4 replication cycles in naïve porcine alveolar macrophages (PAM). While co-infection and recombination frequencies remained low in PAM, they increased sharply between the second and fourth viral cycles. Infection multiplicity and synchronicity significantly influenced ASFV recombination frequency, which correlated positively with genetic distance. Crossing the ASFV/Lv17/WB/Rie1 mutants yielded a saturation curve resembling the Haldane mapping function, plateauing at ~20% recombination frequency at 160 kbp. Crossing less homologous strains (ASFV/Lv17/WB/Rie1 × ASFV/NHV/P68) reduced the recombination frequency; however, at large marker distances (140 kbp), it approached values (0.18) observed in homologous strains. A novel mathematical model estimated the average co-infection rate to be 0.47 after three viral cycles. Empirical application of the model revealed that 1 centimorgan translates to ~550–1650 bp, meaning offspring virions from a single cell undergo an average of 1.15–3.45 recombination events. This high recombination rate is consistent with in vivo observations and has critical implications for ASFV prevention and vaccination strategies. Such genomic plasticity can facilitate rapid viral evolution, potentially undermining the efficacy of live-attenuated vaccines globally.
Neonatal calf diarrhea (NCD) is a major contributor to morbidity and mortality in calves. Growing concern about antimicrobial resistance has spurred interest in phytotherapeutic alternatives, particularly polyphenol-rich extracts. Hazelnut skin (HS), a by-product of the confectionery sector, is a sustainable and underutilized source of polyphenols; however, its therapeutic efficacy in calves with NCD remains insufficiently investigated. This randomized, parallel-group clinical trial evaluated the effects of oral HS extract supplementation as an adjunct to standard medical therapy in hospitalized dairy calves with NCD. Sixty female Holstein calves were randomly allocated to a control group (CTRL; n = 30) or a treatment group (HS; n = 30) receiving 10 g/day of HS extract with milk for 5 days. All calves received standardized clinical management, including fluid therapy and antimicrobial administration. Clinical parameters, milk intake, body weight, hospitalization outcomes, sepsis scores, and blood oxidative stress biomarkers were monitored. No significant differences were detected between groups for clinical recovery, fecal score, hydration status, milk intake, weight gain, hospitalization duration, mortality, or oxidative stress markers. Clinical improvement over time was evident in both groups and was attributable to standard therapy. In conclusion, short-term oral supplementation with HS extract did not confer additional clinical or antioxidant benefits when added to conventional treatment in calves with NCD.
As in humans, therapeutic plasma exchange (TPE) has been investigated in veterinary medicine for managing immune-mediated diseases such as canine visceral leishmaniasis (CVL). TPE may improve clinical outcomes by removing circulating immune complexes and excess antibodies; however, its physiological effects must be understood to ensure safety. Given the scarcity of studies in animals, this study evaluated the effects of centrifugation-based TPE in dogs with CVL receiving fresh frozen plasma (FFP) as replacement fluid. Thirty-one dogs with CVL that failed to respond to conventional therapy were allocated to two groups: clinical treatment alone (n = 18) and clinical treatment plus TPE (n = 13). The TPE group underwent one daily session for three consecutive days. Coagulation profiles were assessed before and after each session. A progressive decrease in platelet counts occurred across sessions, but without significant thrombocytopenia or critical alterations in activated partial thromboplastin time (aPTT) or prothrombin time (PT), which remained within reference ranges. Transient hypocalcemia occurred after the first session and was promptly corrected with calcium gluconate supplementation. Fibrinogen levels decreased mildly without clinical evidence of bleeding. Clinical complications were minimal and effectively managed. In conclusion, TPE was well tolerated, producing predominantly transient and controllable effects on hemostasis and overall clinical homeostasis under the conditions of this study.
PAMK are heterogeneous glycans with immunomodulatory potential, but their structural basis and mechanisms in regulating thymic injury remain unclear. Structural characterization revealed that PAMK is mainly composed of arabinose, galactose, rhamnose, galacturonic acid, and glucose, featuring a dominant backbone consisting of →4)-α-D-GalpA-(1→, →4)-β-D-Galp-(1→, →2)-α-L-Rhap-(1→, and →3,4)-α-D-GalpA-(1→ with highly branched side chains. This complex structure provides the biochemical foundation for its bioactivity. To explore its immunoprotective effects, we established a CTX-induced thymic necroptosis model in goslings. CTX markedly disrupted cytokine profiles and activated necroptosis signaling, accompanied by downregulation of novel-miR-2 and upregulation of TRADD and MLKL. Dietary PAMK supplementation significantly restored cytokine homeostasis and inhibited necroptosis pathway activation. Mechanistically, PAMK upregulated novel-miR-2, which negatively regulated TRADD and MLKL expression, thereby attenuating necroptosis signaling. Overexpression of novel-miR-2 suppressed TRADD/MLKL-mediated necroptosis, whereas its inhibition produced the opposite effect. Furthermore, PAMK mitigated oxidative stress and necroptotic cell death induced by TRADD and MLKL overexpression. Collectively, our findings demonstrate that the defined structural features of PAMK underpin its ability to protect against CTX-induced thymic necroptosis by upregulating novel-miR-2 and suppressing the TRADD/MLKL axis. This work links PAMK's composition to its immunoprotective function and highlights its potential as a safe immunoregulatory agent in poultry.
While dogs' behavioural indicators of psychological stress are widely used, their function as reliable stress markers remains debated. Using the Strange Situation Procedure (SSP) as a social-stress paradigm, we evaluated contextual correlates of dog behaviours to explore their validity as stress indicators. In 64 dogs, behaviours commonly used as stress markers were video-coded and analysed via GLMMs to investigate differences across test episodes and attachment styles (secure, anxious, avoidant), while salivary cortisol, oxytocin, and rectal temperature provided a physiological framework. Episode had significant effects on shaking (p < 0.05), lip-licking (p < 0.001), paw-lifting (p < 0.001), whining (p < 0.001), head-turning (p < 0.001), sniffing the door (p < 0.001), and locomotion (p < 0.001), while attachment style significantly affected head-turning only (p = 0.004). Our findings suggest that the observed behaviours were not expressed linearly in relation to expected stress levels across the SSP episodes. This lack of linearity, combined with the divergent physiological correlates found across attachment groups-for instance, oxytocin correlated positively with lip-licking in securely attached dogs (p < 0.05), but negatively in anxious ones (p < 0.05) -, suggests that these behaviours do not function as simple stress indicators. Rather, their meaning appears to be contingent upon the individual's internal state and the socio-environmental context. We discuss our results in light of an alternative semiotic interpretative framework.
