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Therapeutically, targeting the pro- and anti-apoptotic proteins has been one of the major approaches behind devising strategies to combat associated diseases. Human high-temperature requirement serine protease A2 (hHtrA2), which induces apoptosis through both caspase-dependent and independent pathways is implicated in several diseases including cancer, ischemic heart diseases, and neurodegeneration, thus making it a promising target molecule. In the recent past, the canine model has gained prominence in the understanding of human pathophysiology that was otherwise limited to the rodent system. Moreover, canine models in cancer research provide an opportunity to study spontaneous tumors as their size, lifespan, and environmental exposure are significantly closer to that of humans compared with laboratory rodents. Therefore, using HtrA2 as a model protein, comparative analysis has been done to revisit the hypothesis that canines might be excellent models for cancer research. We have performed evolutionary phylogenetic analyses that confirm a close relationship between canine and human HtrA2s. Molecular modeling demonstrates structural similarities including orientation of the catalytic triad residues, followed by in silico docking and molecular dynamics simulation studies that identify the potential interacting partners for canine HtrA2 (cHtrA2). In vitro biophysical and protease studies depict similarities in interaction with their respective substrates as well as transient transfection of cHtrA2 in mammalian cell culture shows induction of apoptosis. This work, therefore, promises to open a new avenue in cancer research through the study of spontaneous cancer model systems in canines., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Canine parvovirus disease (CPVD) is one of the most common causes of viral diarrhea in dogs. The disease has a mortality rate of up to 90% if left untreated, and can cause gastroenteritis, vomiting, mucoid/hemorrhagic diarrhea, lymphopenia and even immunosuppression. Based on the effects of canine parvovirus type 2 (CPV-2) on the immune system, we investigated the effects of the CPV-2 on hematological indices and the expression of certain immune molecules in blood leukocytes of CPV-positive and non-CPV dogs. The dogs were (para)clinically evaluated, and their disease status was confirmed by antigen rapid detection kits and polymerase chain reaction (PCR). To elucidate the nature of the immunosuppression seen in CPVD, we investigated the expression of caspases 3/7 and 9, and some lectin family molecules such as galectin-1 (important in viral adhesion) and Mincle (macrophage‑inducible C‑type lectin receptor), in blood leukocytes using reverse transcription quantitative PCR (RT-qPCR). We observed remarkable lymphopenia, lower Hb concentration and higher red blood cell distribution width (RDW) value in CPV-positive dogs. No significant changes in expression of caspases 3/7 and 9 were detected, but galectin-1 and Mincle showed remarkable down and up-regulation, respectively. This proves that the immunosuppression seen in most CPVD is not caused by lymphocyte apoptosis in blood, and that Mincle is partially involved in the immune response to CPV-2. The observed changes in galectin-1 and Mincle may be a defense mechanism against parvovirus by potentially preventing the parvovirus from adhering to the cells. Further research is, nonetheless, needed to elucidate the possible roles of these molecules in CPV-2 pathogenesis and the immune system's response to parvovirus., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The spatial organization of biophysical and biochemical cues in the extracellular matrix (ECM) in concert with reciprocal cell-cell signaling is vital to tissue patterning during development. However, elucidating the role an individual microenvironmental factor plays using existing in vivo models is difficult due to their inherent complexity. In this work, we have developed a microphysiological system to spatially pattern the biochemical, biophysical, and stromal cell composition of the ECM along an epithelialized 3D microchannel. This technique is adaptable to multiple hydrogel compositions and scalable to the number of zones patterned. We confirmed that the methodology to create distinct zones resulted in a continuous, annealed hydrogel with regional interfaces that did not hinder the transport of soluble molecules. Further, the interface between hydrogel regions did not disrupt microchannel structure, epithelial lumen formation, or media perfusion through an acellular or cellularized microchannel. Finally, we demonstrated spatially patterned tubulogenic sprouting of a continuous epithelial tube into the surrounding hydrogel confined to local regions with stromal cell populations, illustrating spatial control of cell-cell interactions and signaling gradients. This easy-to-use system has wide utility for modeling three-dimensional epithelial and endothelial tissue interactions with heterogeneous hydrogel compositions and/or stromal cell populations to investigate their mechanistic roles during development, homeostasis, or disease.

The wet noses of dogs and other mammals are attributed to polygonal arrays of fluid-retaining grooves thought to aid in thermoregulation, chemosensation, and even hunting. A new study reveals the mechanical basis of their morphogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The glabrous skin of the rhinarium (naked nose) of many mammalian species exhibits a polygonal pattern of grooves that retain physiological fluid, thereby keeping their nose wet and, among other effects, facilitating the collection of chemosensory molecules. Here, we perform volumetric imaging of whole-mount rhinaria from sequences of embryonic and juvenile cows, dogs, and ferrets. We demonstrate that rhinarial polygonal domains are not placode-derived skin appendages but arise through a self-organized mechanical process consisting of the constrained growth and buckling of the epidermal basal layer, followed by the formation of sharp epidermal creases exactly facing an underlying network of stiff blood vessels. Our numerical simulations show that the mechanical stress generated by excessive epidermal growth concentrates at the positions of vessels that form rigid base points, causing the epidermal layers to move outward and shape domes-akin to arches rising against stiff pillars. Remarkably, this gives rise to a larger length scale (the distance between the vessels) in the surface folding pattern than would otherwise occur in the absence of vessels. These results hint at a concept of "mechanical positional information" by which material properties of anatomical elements can impose local constraints on an otherwise globally self-organized mechanical pattern. In addition, our analyses of the rhinarial patterns in cow clones highlight a substantial level of stochasticity in the pre-pattern of vessels, while our numerical simulations also recapitulate the disruption of the folding pattern in cows affected by a hereditary disorder that causes hyperextensibility of the skin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: The objective of this study was to describe the clinical features, treatments and outcomes of dogs with iron ethylenediaminetetraacetate (EDTA) molluscicide ingestion. We aimed to identify potential predictors of serum iron concentration >54 μmol/L; a cut-off recommended for initiating treatment of human iron toxicosis., Methods: Medical records across four veterinary hospitals (2013-2019) for dogs with known or suspected iron EDTA molluscicide ingestion were reviewed for signalment, clinical signs, clinicopathological data, treatment regimens and outcomes. Clinical signs of dogs with serum iron concentrations either below or above a cut-off of 54 μmol/L were compared using Fisher's exact test. Association between serum iron and urine discolouration was tested using logistic regression., Results: Seventy-three dogs were included. The most frequent characteristic was abnormal faeces (n = 47, 64%) as determined by rectal examination, with more dogs that had serum iron >54 μmol/L showing this clinical sign (24/35), compared to dogs with serum iron <54 μmol/L (4/13) (P = 0.025). Clinicopathologic abnormalities included increased base excess (n = 20/49, 20%), hyperglycaemia (n = 13/57, 23%) and hyperlactataemia (n = 12/57, 21%). Fifty-four dogs received deferoxamine chelation (74%), during which 25/42 (60%) dogs had discoloured urine despite all dogs having subsequent iron concentrations <54 μmol/L. Admission serum iron concentration was associated with urine discolouration during hospitalisation (odds ratio 1.046, 95% confidence interval 1.008-1.107, P = 0.011). Sixty-nine dogs (95%) survived to discharge, with 10 dogs (14%) transferred to another veterinarian., Conclusions: Iron EDTA molluscicide ingestion caused predominantly gastrointestinal clinical signs, and abnormal faeces, as well as urine discolouration during chelation therapy, which may indicate an elevation of serum iron concentration on presentation. Outcome was excellent., (© 2024 Australian Veterinary Association.)

Background: The 1986 disaster at the Chornobyl Nuclear Power Plant released massive amounts of radioactive material into the local environment. In addition to radiation, remediation efforts and abandonment of military-industrial complexes contributed to contamination with heavy metals, organics, pesticides and other toxic chemicals. Numerous studies have evaluated the effects of this contamination on the local ecology. However, few studies have reported the effect of this contamination on vector-borne pathogens and their hosts. In this manuscript, we characterize tick-borne pathogen presence at two sample locations within the Chornobyl Exclusion Zone, one at the Nuclear Power Plant (NPP) and another 16 km away in Chornobyl City (CC)., Methods: Ticks and whole-blood samples were collected from free-breeding dogs captured at the NPP and CC. Endpoint PCR and quantitative PCR were used to identify tick species and to assess the presence of specific tick-borne pathogens, including Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Babesia spp., Bartonella spp., Francisella tularensis and general Anaplasmataceae. A droplet digital PCR assay was developed for Babesia canis and A. phagocytophilum to evaluate their presence in dogs from the two populations. Pathogen prevalences between the two sample populations were compared by calculating Z-scores., Results: Ticks were identified as Ixodes ricinus (n = 102) and Dermacentor reticulatus (n = 4). Overall, 56.9% of I. ricinus ticks were positive for at least one pathogen. A significantly higher prevalence of A. phagocytophilum and B. burgdorferi was found in ticks at the NPP (44.0% and 42.0%, respectively) compared to CC (23.1% and 19.2%, respectively). Babesia spp. (including B. canis and B. caballi) were detected in 8.8% ticks at similar proportions for both populations. Interestingly, we found a significantly lower level of A. phagocytophilum in dogs at the NPP (1.8%) than in dogs at CC (11.7%). In total, 24.3% of dogs were positive for B. canis, evenly distributed across the two populations., Conclusions: The results of this study show contrasting pathogen prevalence in both ticks and dogs at the NPP and CC, which may reflect the differential exposures at the two locations. This work adds an important new component to our understanding of the consequences of prolonged exposure to environmental contamination on the wildlife and ecology within the Chornobyl Exclusion Zone., Competing Interests: Declarations Ethics approval and consent to participate The Chornobyl Nuclear Power Plant authorities under the supervision of licensed veterinarians and veterinary technicians conducted the ethical review and provided permission for the animal study. A letter of support detailing the cooperation between the Clean Futures Fund and the Chornobyl NPP was signed by the NPP acting general director V.A. Seyda and can be provided by TAM upon request. Approval for access to the exclusion zone was handled and granted by the Exclusion Zone Authority. Samples collected for this study were gathered adventitiously while animals were being treated by the medical program and as such are exempt from IACUC approval. Consent for publication Not applicable. Competing interests BAQ receives partial salary support from IDEXX Laboratories, Inc and has been a paid consultant during the development and validation of rapid assays developed by IDEXX Laboratories for veterinary medical applications. The other authors have no competing interests to declare., (© 2024. The Author(s).)

Influenza remains a serious issue for public health and it's urgent to discover more effected drugs against influenza virus. Rhamnocitrin, as a flavonoid, its effect on influenza virus infection remains poorly explored. In this study, rhamnocitrin showed antiviral effect and anti-apoptosis on influenza virus A/Aichi/2/1968 (H3N2) in MDCK cells and A549 cells. In addition, molecular docking revealed that rhamnocitrin have good binding activity with the target proteins cGAS and STING, molecular dynamic simulation and surface plasmon resonance showed that rhamnocitrin could form a stable complex with the above proteins. Moreover, the qPCR and western blot assays further verified that rhamnocitrin could reduce type I IFN and proinflammatory cytokines production by inhibiting the cGAS/STING pathway. Taken together, the results suggest that rhamnocitrin could be a potential anti-viral agent against influenza., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Dirofilaria sp. "hongkongensis" is a putative Dirofilaria species, initially identified in subcutaneous nodules in humans in Hong Kong and in other South and Southeast Asian regions. While it differs genetically from the better-known zoonotic species, Dirofilaria repens and Dirofilaria immitis, information on the lesions caused by Dirofilaria sp. "hongkongensis" in the hosts as well as on its biology is scarce. This study documents for the first time the presence of this filarioid nematode in subcutaneous nodules in dogs and cats in Hong Kong, where it was originally described in human patients, therefore providing evidence for the zoonotic nature of this parasite., Methods: Records of Veterinary Diagnostic Laboratory of City University of Hong Kong were searched between 2019 and 2024 for histological reports of possible filarioid-associated lesions. Tissue samples were collected by excisional surgical biopsy and processed with routine paraffin techniques. Selected slides were stained using various staining techniques [i.e., hematoxylin and eosin, periodic acid-Schiff (PAS), Grocott methenamine silver (GMS) or Ziehl-Neelsen (ZN) and Gram stain]. DNA from formalin-fixed paraffin-embedded tissue were extracted, submitted to conventional polymerase chain reaction (cPCR) and sequencing (i.e., cox1 and 12S rRNA genes) and phylogenetic analyzed., Results: A total of five subcutaneous nodules from four cats and one from a dog with histopathology suggestive of filariosis were selected. The presence of Dirofilaria sp. "hongkongensis" was morphologically and molecularly confirmed in one dog and one cat. Both histopathological presentation and phylogenetic analysis enabled classification of this species close to D. repens and within the subgenus Nochtiella. In the remaining three cases, one showed histological evidence of aberrant nematode migration, while non-parasitic causes were identified in the other two., Conclusions: This study provides the first evidence of Dirofilaria sp. "hongkongensis" in subcutaneous nodules in cats and dogs. The histology of clinical lesions of this filarioid species herein described is closely related to those caused by D. repens. Overall, this species should be considered in differential diagnoses of subcutaneous lesions in both animals and humans in the region., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Visceral leishmaniasis (VL) is a zoonotic disease caused by Leishmania infantum and transmitted by the sand fly Lutzomyia longipalpis. Dogs are the major domestic reservoir of L. infantum. To prevent the spread of the disease, dog collars impregnated with 4% deltamethrin have been effectively used in VL endemic areas. However, this approach may contribute to the emergence of insecticide resistance in sand flies. Therefore, it is important to characterize the susceptibility of different populations of Lu. longipalpis to deltamethrin in areas where insecticide-impregnated dog collars are used., Methods: Six field sand fly populations from Brazil were exposed to deltamethrin in CDC bottle bioassays at the diagnostic doses (DD) of 21.9 μg/bottle and 30 μg/bottle. For the dose-response (DR) experiments, doses of 1, 3, 5, 7, 9 and 11 μg/bottle of deltamethrin were used to impregnate bottles; control group bottles were impregnated with acetone only. Each bottle contained an average of 20 sand flies, both male and female, and they were exposed to either deltamethrin or acetone for 60 min., Results: Based on the DD of 21.9 μg/bottle, three populations were susceptible to deltamethrin. In contrast, two populations collected from the states of Ceará and Minas Gerais exhibited mortality rates of 94.9% and 95.7%, indicating possible resistance, and one population from the state of Ceará showed resistance, with a mortality rate of 87.1%. At the DD of 30 μg/bottle, two populations from the states of Ceará and Piauí showed possible resistance, while the other four populations were susceptible. The resistance ratio (RR 50 ) ranged from 2.27 to 0.54, and RR 95 ranged from 4.18 to 0.33, indicating a low resistance intensity., Conclusions: This study established a DD for Lu. longipalpis using the CDC bottle bioassay. We found that Lu. longipalpis populations in three Brazilian states where insecticide-impregnated dog collars were used for VL control were susceptible to deltamethrin. However, one population in Ceará State was classified as resistant to deltamethrin. These results contribute to the current knowledge on sand fly resistance and surveillance, and highlight the need for a better understanding of the resistance mechanisms of Lu. longipalpis in areas where insecticide-impregnated dog collars have been widely used., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The specificity and sensitivity of a simple non-invasive multi-cancer screening method in detecting breast, lung, prostate, and colorectal cancer in breath samples were evaluated in a double-blind study. Breath samples of 1386 participants (59.7% males, median age 56.0 years) who underwent screening for cancer using gold-standard screening methods, or a biopsy for a suspected malignancy were collected. The samples were analyzed using a bio-hybrid platform comprising trained detection canines and artificial intelligence tools. According to cancer screening/biopsy results, 1048 (75.6%) were negative for cancer and 338 (24.4%) were positive. Among the 338 positive samples, 261 (77.2%) were positive for one of the four cancer types that the bio-hybrid platform was trained to detect, with an overall sensitivity and specificity of 93.9% (95% confidence interval [CI] 90.3-96.2%) and 94.3% (95% CI 92.7%-95.5%), respectively. The sensitivity of each cancer type was similar; breast: 95.0% (95% CI 87.8-98.0%), lung: 95.0% (95% CI 87.8-98.0%), colorectal: 90.0% (95% CI 74.4-96.5%), prostate: 93.0% (95% CI 84.6-97.0%). The sensitivity of 14 other malignant tumors that the bio-hybrid platform was not trained to detect, but identified, was 81.8% (95% CI 71.8%-88.8%). Early cancer (0-2) detection sensitivity was 94.8% (95% CI 91.0%-97.1%). This bio-hybrid multi-cancer screening platform demonstrated high sensitivity and specificity and enables early-stage cancer detection., Competing Interests: Declarations Competing interests Milana Avdalimov and Assaf Rabinowicz are SpotitEarly Ltd. employees. Sharon Furman-Assaf and Ronit Shmuel received funding from SpotitEarly Ltd. All other authors have no competing interests to declare., (© 2024. The Author(s).)

The confirmation of a connection between the oral and nasal cavities provides a definitive diagnosis for an oronasal fistula (ONF). However, visual detection is difficult in patients with small fistulas. This study examined whether fluorescein sodium could be used to detect small ONFs. The study included 25 dogs that underwent dental examination and had confirmed periodontal pockets at the Amica Pet Clinic, Japan, between 2020 and 2022. Saline containing fluorescein sodium was injected into the periodontal pocket, termed a fluorescent "water-passing test," followed by detection of fluid discharge from the external naris using an examination light. Of the 25 dogs, 13 showed symptoms consistent with an ONF and of these, 12 were positive and one was negative on the fluorescent water-passing test. Of the 12 asymptomatic dogs, 3 tested positive and 9 tested negative on the fluorescent water-passing test. The fluorescent water-passing test enhanced visibility and facilitated the detection of small volumes of the discharged fluid. The results of this study confirmed the fluorescent water-passing test was useful for the detection of small ONFs., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Liposomal drug delivery systems have revolutionized traditional cytotoxic drugs. However, the relative instability and toxicity of the existing liposomal drug delivery systems compromised their efficacy. Herein, we present Rg3-lipo, an innovative drug delivery system using a glycosyl moiety-enriched ginsenoside (Rg3). This system is distinguished by its glycosyl moieties exposed on the liposomal surface. These moieties imitate human cell membranes to stabilize and evade phagocytic clearance. The Rg3-lipo system loaded with paclitaxel (PTX-Rg3-lipo) demonstrated favorable bioavailability and safety in Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys. With its glycosyl moieties recognizing tumor cells via the glucose transporter Glut1, PTX-Rg3-lipo inhibited gastric, breast, and esophageal cancers in human cancer cell lines, tumor-bearing mice, and patient-derived xenograft models. These glycosyl moieties selectively targeted myeloid-derived suppressor cells (MDSCs) through the glucose transporter Glut3 to attenuate their immunosuppressive effect. The mechanism study revealed that Rg3-lipo suppressed glycolysis and downregulated the transcription factors c-Maf and Mafb overcoming the MDSC-mediated immunosuppressive microenvironment and enhancing PTX-Rg3-lipo's antitumor effect. Taken together, we supply substantial evidence for its advantageous bioavailability and safety in multiple animal models, including nonhuman primates, and Rg3-lipo's dual targeting of cancer cells and MDSCs. Further investigation regarding Rg3-lipo's druggability will be conducted in clinical trials.

Cerebrospinal fluid (CSF) circulation diseases, such as hydrocephalus and syringomyelia, are common in small-breed dogs. In human patients with CSF circulation diseases, time-spatial labeling inversion pulse (time-SLIP) sequence performed to evaluate CSF flow before and after treatment allows visualization of the restoration of CSF movement. However, studies evaluating CSF flow using the time-SLIP method in small-breed dogs are limited. Therefore, the present study aimed to evaluate intracranial CSF flow on time-SLIP images in small-breed dogs with idiopathic epilepsy, as an alternative model to healthy dogs. Time-SLIP images were obtained at two sites: 1) the mesencephalic aqueduct (MA) area (third ventricle, MA, and brain-base subarachnoid space [SAS]) and 2) the craniocervical junction area (fourth ventricle, brainstem, and cervical spinal cord SAS) to allow subsequent evaluation of the rostral and caudal CSF flow using subjective and objective methods. In total, six dogs were included. Caudal flow at the MA and brain-base SAS and rostral flow in the brainstem SAS were subjectively and objectively observed in all and 5/6 dogs, respectively. Objective evaluation revealed that a significantly smaller movement of the CSF, assessed as the absence of CSF flow by subjective evaluation, could be detected in some areas. In small-breed dogs, the MA, brain-base, and brainstem SAS would be appropriate areas for evaluating CSF movement, either in the rostral or caudal flows on time-SLIP images. In areas where CSF movement cannot detected by subjective methods, an objective evaluation should be conducted.

Companion animals have the potential to greatly enhance the physical and mental health of humans, thus leading to an increased focus on the interactions between humans and pets. Currently, the inappropriate and excessive utilization of antimicrobial agents has become prevalent in veterinary clinical practice for pets. This antibiotic contamination phenomenon has a profound impact on the enrichment of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) in pets. However, the pet-associated resistome, especially the novel ARGs in pets, represents a relatively neglected area. In this study, we successfully constructed a total of 12 libraries using the functional metagenomics approach to assess the diversity of ARGs in pet cats and dogs from four pet hospitals. Through the integration of functional screening and high-throughput sequencing, a total of 122 antibiotic resistance determinants were identified, of which 15 were classified as putative novel ARGs originating from five classes. Functional assessment demonstrated that 6 novel ARGs including one β-lactam, two macrolides, two aminoglycosides, and one rifamycin (RIF), namely bla PF , erm PF , msr PF , aac(6') PF , aph(3') PF , and arr PF , exhibited functionally activity in conferring bacterial phenotypic resistance by increasing the minimum inhibitory concentrations (MICs) with a 4- to 128-fold. Genetic context analysis demonstrated that, with the exception of aac(6') PF and arr PF , the remaining four novel ARGs were found adjacent to mobile genetic elements (MGEs) including IS elements or transposases, which provided a prerequisite for horizontal transfer of these novel ARGs, thereby offering an explanation for their detection in diverse samples collected from various sampling sites. The current study has unveiled the significant role of cat and dog feces as one source of reservoirs of diverse novel ARGs, while also highlighting the potential adverse consequences of their further spread to medically significant pathogens and human commensal organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Sangju Cold Granule (SJCG) is a classical traditional Chinese medicine (TCM) prescription described in "Item Differentiation of Warm Febrile Diseases". Historically, SJCG was employed to treat respiratory illnesses. Despite its popular usage, the alleviating effect of SJCG on influenza A virus infection and its mechanisms have not been fully elucidated., Aim of the Study: Influenza is a severe respiratory disease that threatens human health. This study aims to assess the therapeutic potential of SJCG and the possible molecular mechanism underlying its activity against influenza A virus in vitro and in vivo., Materials and Methods: Ultrahigh-performance liquid chromatography (UPLC)-Q-Exactive was used to identify the components of SJCG. The 50% cytotoxic concentration of SJCG in MDCK and A549 cells were determined using the CCK-8 assay. The activity of SJCG against influenza A virus H1N1 was evaluated in vitro using plaque reduction and progeny virus titer reduction assays. RT-qPCR was performed to obtain the expression levels of inflammatory mediators and the transcriptional regulation of RIG-I and MDA5 in H1N1-infected A549 cells. Then, the mechanism of SJCG effect on viral replication and inflammation was further explored by measuring the expressions of proteins of the RIG-I/NF-kB/IFN(I/III) signaling pathway by Western blot. The impact of SJCG was explored in vivo in an intranasally H1N1-infected BALB/c mouse pneumonia model treated with varying doses of SJCG. The protective role of SJCG in this model was evaluated by survival, body weight monitoring, lung viral titers, lung index, lung histological changes, lung inflammatory mediators, and peripheral blood leukocyte count., Results: The main SJCG chemical constituents were flavonoids, carbohydrates and glycosides, amino acids, peptides, and derivatives, organic acids and derivatives, alkaloids, fatty acyls, and terpenes. The CC 50 of SJCG were 24.43 mg/mL on MDCK cells and 20.54 mg/mL on A549 cells, respectively. In vitro, SJCG significantly inhibited H1N1 replication and reduced the production of TNF-α, IFN-β, IL-6, IL-8, IL-13, IP-10, RANTES, TRAIL, and SOCS1 in infected A549 cells. Intracellularly, SJCG reduced the expression of RIG-I, MDA5, P-NF-κB P65 (P-P65), P-IκBα, P-STAT1, P-STAT2, and IRF9. In vivo, SJCG enhanced the survival rate and decreased body weight loss in H1N1-infected mice. Mice with H1N1-induced pneumonia treated with SJCG showed a lower lung viral load and lung index than untreated mice. SJCG effectively alleviated lung damage and reduced the levels of TNF-α, IFN-β, IL-6, IP-10, RANTES, and SOCS1 in lung tissue. Moreover, SJCG significantly ameliorated H1N1-induced leukocyte changes in peripheral blood., Conclusions: SJCG significantly reduced influenza A virus and virus-mediated inflammation through inhibiting the RIG-I/NF-kB/IFN(I/III) signaling pathway. Thus, SJCG could provide an effective TCM for influenza treatment., Competing Interests: Declaration of Competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Leptospirosis is a globally important re-emerging zoonotic disease affecting humans and many animal species including dogs. The present cross-sectional study aimed to diagnose acute leptospirosis among 210 suspected dogs using microscopic agglutination test (MAT) and polymerase chain reaction (PCR). Further, epidemiological risk factors were analyzed by univariate analysis and multivariate binomial logistic regression analysis. Out of the 210 dogs, anti-leptospiral antibody was detected in 123 (58.57%; 95% CI: 51.6-65.3) dogs by MAT (cut-off titer- ≥1:100), among which 67 (54.47%; 95% CI: 45.3-63.4) were seropositive to more than one serogroup. The predominant serogroups were Australis and Autumnalis in the study region. Whereas, acute leptospirosis was established in 49 (23.33%, 95% CI: 17.9-29.8) dogs based on ≥ 1:800 titer in unvaccinated dogs and ≥ 1:1600 titer in vaccinated dogs. The predictive risk factors of acute canine leptospirosis were adult dogs (1-5 years) (p=0.001), north-east monsoon season (p=0.011), outdoor management (p=0.047), history of rodent exposure (p=0.001) and history of contact with wet soil or stagnant water (p=0.046). Among 49 acutely infected dogs, thirteen dogs were positive for urine PCR and one dog was positive for both urine as well as plasma PCR. Positive PCR amplicons were identified as Leptospira interrogans based on secY gene sequencing and phylogenetic analysis. This study enlightened about the occurrence of acute leptospirosis among suspected dogs with the above important predictive risk factors, which should be taken into consideration while history taking so that proper treatment strategies can be adopted for early recovery of the animal., Competing Interests: Declaration of Competing Interest ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Objective: A promising strategy to increase population physical activity is through promotion of dog walking. Informed by multi-process action control and nascent dog-walking theory, we examined the effectiveness of a 3-month technology-based (dog tracker) 2-arm randomised controlled dog-walking intervention to increase dog-owner daily physical activity in the general community in Sydney, Australia., Results: 37 participants were allocated to the intervention group (mean age = 43.2 [SD 11.9]) and 40 to the control group (mean age = 42.3 [SD 11.9]). Both groups averaged more than 10,500 steps/day at baseline. There was no evidence of within- or between-group physical activity differences across timepoints. The results remained consistent after exclusion of participants who had data collected during COVID-19 lockdowns. Compared with baseline, both groups had significant increases in sedentary time during the post-intervention, and 6 month follow-up. The absence of significant differences between-group physical activity differences may be attributable to the ceiling effect of both groups already being sufficiently active. These results provide useful guidance to future studies intended to assess the efficacy of technology-based dog-walking interventions. Future dog-walking interventions should specifically target physically inactive dog owners., Trial Registration: ACTRN12619001391167 (10/10/2019); Retrospectively registered., Competing Interests: Declarations Ethics approval and consent to participate Ethical approval was obtained from the University of Sydney Human Research Ethics Committee (2016/921) and the Animal Ethics Committee (2017/1134). The study was registered with the Australian New Zealand Clinical Trials Registry (https://www.anzctr.org.au/). All participants provided informed written consent. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Over the past decade, Lyme and other tick-borne diseases have expanded into urban areas, including Staten Island, New York. While Lyme disease is often researched with a focus on human risk, domestic pets are also at risk of contracting the disease. The present study aims to describe differences in tick exposure, knowledge, attitude, and practices (KAP) between pet owners and non-owners, and to understand preventive strategies practiced by pet owners for themselves and their pets. We conducted KAP surveys via phone in 2020 and via face-to-face interviews in 2021, and we analyzed unique responses from 364 households on Staten Island. Pet owners were more likely to have ever found a tick on themselves or their household members (63%) than non-owners (46%) (p<0.001). Among pet owners, those who owned dogs (dog-only or both dog and cat owners) were more likely to have ever found a tick on their pets than cat-only owners (p<0.001). Compared with non-pet owners, pet owners were more likely both to know that ticks transmit Lyme disease (p<0.001) and to avoid gardening to reduce their tick exposure (p = 0.032), but they were less likely to wear protective clothing or adjust clothing (p = 0.013). Compared with cat owners who had never found a tick on their cats, cat owners who had ever found a tick on their cats were more likely to let their cats go outside (p<0.001). However, reported preventive measures on cats did not differ between pet owners who did and did not report tick exposure. The results indicate that encouraging pet owners to engage in preventative measures, both to protect themselves and their pets, is a potential avenue for healthcare providers and veterinarians to reduce risks from ticks., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Shape bias, the tendency to link the meaning of words to the shape of objects, is a widely investigated phenomenon, but the extent to which it is linked to vocabulary acquisition and/or to tool using is controversial. Understanding how non-human animals generalize the properties of objects can provide insights into the evolutionary processes and cognitive mechanisms that influence this bias. We investigated object generalization in dogs, focusing on their tendency to attend to shape or texture in a two-way choice task. We analyzed data of thirty-five dogs that were successfully trained to discriminate a target object among distractors and retrieve it to their owner. In subsequent testing, dogs chose between objects similar to the target in either shape or texture. Results showed that dogs first approached objects of similar shape, but then predominantly chose objects of similar texture. This suggests a reliance on visual cues for initial assessment and tactile cues for final discrimination. Our findings highlight the influence of perceptual modalities on object generalization in dogs, showing that, before object manipulation, they seem to show a shape bias but, once they make physical contact with the objects, they rather rely on texture to generalize. This study contributes to our understanding of generalization in a non-verbal and non-tool using species and opens avenues for comparative investigations into the relationship between vocabulary acquisition, tool using and biases in object generalization., Competing Interests: Competing interests The authors declare no competing interests. Informed consent Owners gave their informed consent for participating with their dogs in the study., (© 2024. The Author(s).)

MDM2 has been pursued as an attractive therapeutic target for human cancers. Herein, we describe our discovery of MD-265 as a promising PROTAC MDM2 degrader and extensive in vitro and in vivo evaluations of its therapeutic potential and mechanism of action. MD-265 effectively depleted MDM2 protein in cancer cells at concentrations as low as 1 nM, leading to strong activation of p53 in cancer cells carrying wild-type p53. It selectively inhibited the growth of wild-type p53 leukemia cell lines and showed no activity in mutated p53 lines. MD-265 achieved persistent tumor regression in a leukemia xenograft model without causing any signs of toxicity and dramatically improved survival of mice in a disseminated leukemia model even with a weekly administration. MD-265 displayed an excellent intravenous PK profile in mice, rats, and dogs. MD-265 is a promising MDM2 degrader for advanced preclinical development for the treatment of human cancers.

Hydrocarbon-determined shake-flask measurements have demonstrated great utility for optimizing lipophilicity during early drug discovery. Alternatively, chromatographic methods confer reduced experimental error and improved handling of complex mixtures. In this study, we developed a chromatographic approach for estimating hydrocarbon-water shake-flask partition coefficients for a variety of macrocyclic peptides and other bRo5 molecules including PROTACs. The model accurately predicts experimental shake-flask measurements with high reproducibility across a wide range of lipophilicities. The chromatographic retention times revealed subtle conformational effects and correlated with the ability to sequester hydrogen bond donors in low dielectric media. Estimations of shake-flask lipophilicity from our model also accurately predicted trends in MDCK passive cell permeability for a variety of thioether-cyclized decapeptides. This method provides a convenient, high-throughput approach for measuring lipophilic permeability efficiency and predicting passive cell permeability in bRo5 compounds that is suitable for multiplexing pure compounds or investigating the properties of complex library mixtures.

Inhibition of integrin αvβ6 is a promising approach to the treatment of fibrotic disease such as idiopathic pulmonary fibrosis. Screening a small library combining head groups that stabilize the bent-closed conformation of integrin αIIbβ3 with αv integrin binding motifs resulted in the identification of hit compounds that bind the bent-closed conformation of αvβ6. Crystal structures of these compounds bound to αvβ6 and related integrins revealed opportunities to increase potency and selectivity, and these efforts were accelerated using accurate free energy perturbation (FEP+) calculations. Optimization of PK parameters including permeability, bioavailability, clearance, and half-life resulted in the discovery of development candidate MORF-627, a highly selective inhibitor of αvβ6 that stabilizes the bent-closed conformation and has good oral PK. Unfortunately, the compound showed toxicity in a 28-day NHP safety study, precluding further development. Nevertheless, MORF-627 is a useful tool compound for studying the biology of integrin αvβ6.

Leishmaniasis are zoonosis widely spread in Brazil, caused by the protozoan of the genus Leishmania, which includes several species. The disease manifests itself in the visceral or tegumentary form, and the main reservoir is the dogs. Manaus is the largest city in the Brazilian Amazon region, and despite the importance of the municipality, practically nothing is known about leishmaniosis in humans and animals. The objective of this study was to evaluate the occurrence and risk factors associated with the presence of Leishmania infantum in domiciliated dogs from Manaus. Molecular (polymerase chain reaction) and serological (immunofluorescent antibody test) methods were used as an indication of the circulation of the parasite. Blood samples for 154 domiciled dogs were obtained, and prevalence ratio and analysis of the variables were performed. Serum antibodies anti-Leishmania spp. were detected in 20.8% (95% CI: 14.4%-27.2%). Access to the street and zone of residence (P < 0.01) were associated with higher seropositivity. Molecular diagnosis for L. infantum detected positivity in 60 (39%) of the 154 (95% CI: 31.3%-46.7%) animals, and the variables street access, contact with dogs, and zone of residence were associated with higher frequencies of positivity (P < 0.05). Both serology and molecular diagnosis detected positive dogs in the municipality. This is the first description of the circulation of L. infantum infecting dogs in Manaus. As the municipality is classified as nonendemic, studies of isolation and characterization of the isolate must be done urgently., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

In this study, a multiplex PCR method was developed for the detection of four diarrhea-associated viruses of canines, including canine bocavirus (CBoV), canine circovirus (CCV), torque teno canis virus (TTCV), and canine kobuvirus (CKV). Four pairs of compatible primers, one specific for each virus, were designed based on conserved sequences. After optimization of parameters such as primer concentration and annealing temperature in single and multiple amplifications, four specific fragments were amplified simultaneously with high sensitivity and specificity in one PCR reaction. The fragments amplified were 165 bp (CBoV), 345 bp (CCV), 506 bp (TTCV), and 666 bp (CKV) in length. The sensitivity of this one-step multiplex PCR is about 10 times lower than that of regular singleplex PCR. There was no cross-reaction with the canine pathogens canine parvovirus (CPV), canine distemper virus (CDV), or canine coronavirus (CCoV). Testing of canine fecal samples from China using the multiplex PCR assay revealed the presence of CBoV, CCV, TTCV, and CKV in 10.1%, 6.2%, 2.8%, and 1.7% of the samples, respectively. The results of multiplex PCR agreed with the singleplex PCR results with a coincidence rate of 100%. In addition, the complete genome sequences of the viruses in three CKV-positive samples were determined and found to be 95.7 - 96.6% identical to the reference strain US-PC0082 and genetically more distant from other animal kobuvirus. The multiplex PCR method established in this study is convenient, with high specificity and sensitivity, which will be helpful for the rapid differential diagnosis of CBoV, CCV, TTCV, and CKV infections., Competing Interests: Declarations Ethical approval All animal experiments conformed to the guidelines and regulatory requirements established by the Animal Care and Use Committee of Qingdao Agricultural University, Shandong Province, China. The owners’ consent was obtained to collect samples from diarrheic dogs. Conflict of interest All authors declare no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Purpose of Review: Allergic rhinitis and asthma morbidity has been linked to indoor allergen exposure. Common indoor allergens include dust mites, cats, dogs, rodents, and cockroaches. These allergens are ubiquitous and often difficult to remove from the home, making long-lasting reduction strategies difficult to achieve. Identifying strategies for reducing the presence of indoor allergens in homes could be utilized to decrease allergic disease burden, improve symptomology, reduce healthcare costs, and improve patients' quality of life., Recent Findings: Studies have yielded mixed results with regard to specific environmental control measures in reducing indoor allergen levels and in improving clinical outcomes of allergic disease. In this review, we assess the available evidence of the effectiveness of environmental control measures in reducing indoor allergens and the potential clinical impact of these measures., Competing Interests: Declarations Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors. Conflict of Interest TLG receives research support from NIAID. RAW receives research support from NIAID, Aimmune, ALK, DBV, FARE, Genentech, Novartis, and Siolta. Competing Interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

There are no regulations for the flyball box angulation, which ranges from 45° to 89°. As such at present, the box turn is deemed to represent the greatest injury risk to competitors. The aim of this study was to understand the influence of box angle on kinematic variables during a flyball turn, by comparing dogs turning on three different angulations of flyball box (45°, 60° and 83°) to allow for recommendations to be made regarding the most appropriate box design in terms of limiting risk of injury across the sport, to increase both wellbeing and safety for competitors. Turning on a 45° box generates significantly more flexion in the forelimbs and carpus, whereas turning on an 83° box generates greater degrees of extension in the elbow, shoulder, hock and stifle. What our 3D analysis has shown is that the relationship between box angle and the physical demands placed on the dog are complex, and related mainly to asymmetrical nature of the sport, and as such no one angle may be more or less suitable for training and competition, but the 60° seems to be a mid-ground, whereas direction of turn may be fundamental in generating the potential for injury., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Hypertriglyceridemia (HTG) is influenced by multiple genetic and environmental factors. Spontaneous, idiopathic HTG is common in the Miniature Schnauzer dog and presumed to have a strong genetic influence in this breed. To define genes that are differentially expressed in dogs with HTG, we performed RNA sequencing on peripheral blood of 13 Miniature Schnauzers with HTG and 18 controls. We identified 110 differentially expressed genes (DEGs). Pathway analysis suggests an ongoing pro-thrombotic, endothelial activation process in dogs with HTG. The gene with the largest fold change (5.4 ± 1.4, Padj = 4.4E-04), SERPINE1, encodes plasminogen activator inhibitor 1 (PAI-1), a known risk factor for atherosclerosis and thrombosis. Other top DEGs, including SHANK3, MMRN1, and FZD7, are involved in endothelial activation. Two of the top DEGs, ARHGAP29 and ARHGAP21, inhibit pro-thrombotic pathways and are potentially protective of disease sequelae. Top DEGs, including SERPINE1 and ARHGAP21, have also been linked to metabolic syndrome or its features (e.g. insulin resistance) in humans and animal models. Our findings indicate that HTG in the Miniature Schnauzer dog has similar features to HTG and metabolic syndrome in humans, highlighting the potential use of the dog as a spontaneous model for further research into the etiology and effects of HTG., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Baker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Owned dogs are often regarded as family members, best friends, or even as substitutes for children. These new roles and their effects on human well-being have received scientific attention, but little is known about how attributing such social functions to dogs might influence the caring behaviour of the owner, which in turn can impact canine welfare. Our online survey of Hungarian dog owners (N = 790) aimed to describe what owners appreciate the most and the least in their dogs and to investigate the relationship between the social and non-social functions of the dog, the characteristics of the owner and the dog, the management practices of the owner, as well as the dog's behaviour, including behaviour problems. We found that most owners appreciated petting and physical contact with their dog (97.6%), followed by 'unconditional love' (93.7%), the dog's beauty (88.4%), and walking with the dog (86.5%). The most frequently mentioned problems were socialisation issues (20.4%) and lack of time (15.9%). Regarding canine behaviour, jumping up (33.2%), chasing animals (28.5%), territoriality (26.7%), and overexcitement (20.9%) were common problems. Almost a quarter of owners rated their dogs as totally obedient and 30.6% as totally safe off-leash. We identified three profiles of dog owners based on the roles they attribute to their dogs. Dogs with both companionship and practical functions (31.1%) were perceived as more obedient and provided higher safety benefits to their owners than others. Dogs regarded as 'companion animals' (19.4%) spent less time with their owners and had older owners than others. Lastly, owners labelled as 'dog parents' (49.5%) were more likely to keep their dog indoors only than others. Importantly, the roles attributed to dogs were not associated with owner-reported canine behaviour problems. We concluded that while the functions humans attribute to dogs are multiple and appear to have direct implications for the dogs' daily lives, these variations in management practices do not necessarily seem to compromise their welfare., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Lyme disease, caused by Borreliella burgdorferi and related species , is a growing health threat to companion animals across North America and Europe. Vaccination is an important preventive tool used widely in dogs living in, or near, endemic regions. In this report, we assessed anti-outer surface protein (Osp) A and anti-OspC antibody responses in B. burgdorferi -infected and -naïve mice (C3H/HeN) after immunization with a murine-optimized single dose of the Lyme disease subunit vaccine, Vanguard crLyme. crLyme is comprised of OspA and an OspC chimeritope-based immunogen designated as CH14. Mice that were infected and immunized developed higher levels of anti-OspC antibodies (Abs) than those infected only or that received one vaccine dose. The anti-OspC Abs that developed in the infected/immunized mice bound to all OspC variants tested ( n = 22), whereas OspC Abs in serum from infected mice bound predominantly to the OspC variant (type A) produced by the infecting B. burgdorferi strain. Consistent with the absence of OspA expression in infected mammals, none of the infected mice developed Abs to OspA and did not develop anti-OspA Abs after single dose immunization. Lastly, serum from infected/immunized mice displayed significantly higher and broader killing activity than serum from non-immunized infected mice. The results of this study demonstrate that a single vaccination of actively infected mice results in strong anti-OspC Ab responses. This study contributes to our understanding of Ab responses to vaccination in actively infected mammals., Competing Interests: R.T.M. is a co-inventor of the crLyme vaccine and receives royalties from the VCU Intellectual Property Foundation. This study was supported, in part, by a grant from Zoetis Petcare. The sponsor played no role in the study design, data interpretation, or manuscript preparation.

Background: Microfilaria (MF) testing is an essential part of canine heartworm diagnostics, and it is recommended by the American Heartworm Society that a MF test be performed in tandem with antigen testing on every dog, every year, regardless of prevention status or history. There are a variety of methods that can be used to detect MF in canine whole blood; however, these methods widely vary in their sensitivities as well as practical factors, including time investment and cost. Additionally, some MF tests offer the advantage of being quantitative or allowing for morphological or molecular species identification, while other tests should only be used qualitatively., Methods: The purpose of this study is to evaluate the quantitative and qualitative performance of MF tests, including the 20 μL count, wet mount, 9 μL and 40 μL hematocrit tubes, thin smear, thick smear, modified Knott test (MKT), and conventional polymerase chain reaction (PCR)., Results: Qualitatively, there was little difference in the performance of the 20 μL count, wet mount, MKT, and PCR. The MKT and PCR are the optimal MF tests, as these perform most reliably for detecting positives even when the MF per milliliter is relatively low, and in most cases, these two methods also allow for species-level confirmation of the identity. However, PCR tends to be a very costly test, and both PCR and MKT require a greater degree of expertise and time investment to perform than other tests. Even the lowest performance tests, including the thin smear and hematocrit tube methods, can reliably detect MF at very high burdens; although, caution should be advised when using low reliability methods, since there is a greater likelihood of failing to identify MF-positive dogs., Conclusions: Microfilaria (MF) testing is an essential part of heartworm diagnosis and screening in dogs, and test selection should balance practical factors such as cost and time investment with the patient's risk of infection based on prevention status and history, clinical signs, and antigen testing results. This approach to MF testing will help minimize cost while avoiding failure to detect MF in infected dogs, especially when MF burden is low., Competing Interests: Declarations Ethics approval and consent to participate The dogs utilized for blood collection were managed according to protocol A2022 04-009, which had prior approval by the University of Georgia Institutional Animal Care and Use Committee. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Dog overpopulation and stray dogs are global issues that are detrimental to public health and animal welfare. Thus, the goal of the current study was to provide alternatives for surgical castration. Therefore, calcium chloride was employed in this study, which might be an option for castration. Ten dogs were divided into two groups of five: a calcium chloride-treated group and a control group. The treated group received a single bilateral intratesticular injection of 1 ml of sterile saline containing calcium chloride dihydrate (CaCl2•2 H2O) at a dose of 20 mg/kg per testicle. While the control group was treated with 1 ml of sterile saline solution, Semen and blood collection, as well as Doppler ultrasonography, were routinely carried out every week on days 0, 7, 14, 21, and 28 in order to evaluate the impact of the injection on semen parameters and testicular blood flow. The testicular volume and echogenicity in the CaCl2-treated group were significantly (P < 0.001) lower in weeks 2 through 4 than in the control group. Furthermore, in canine semen, CaCl2 dramatically decreased the amount, motility, and viability of sperm. When compared to vehicle-control animals, azoospermia was seen 2 weeks after the injection and persisted for the end of the study. The testes of all dogs were surgically removed at 30 days post-injection, and testes were put in 10% neutral buffered formalin for tissue processing. When compared to the control group, the average weight of testes in the chemical groups was dramatically reduced. Significant decreases in spermatogenic processes, necrosis, and degeneration of seminiferous tubules packed with necrotic debris, and fibrosed interstitial tissue, necrosed and calcified Sertoli, and Leydig cells were seen 30 days after CaCl2 injection. There was a significant decrease in testosterone levels compared to day 0 before CaCl2 injection and the control group. From weeks 1 through 4, there was a substantial decrease in both peak systolic velocity (PSV) and end-diastolic velocity (EDV) values (P < 0.001) following a single intratesticular injection of CaCl2. The resistance index (RI) and pulsatility index (PI) showed the opposite tendency. Based on the histopathological and semen evaluations in this investigation, the study concludes that a single intratesticular injection of CaCl2 appears to be a practical and generally applicable approach for chemical sterilization of dogs., Competing Interests: Declarations Ethics approval and consent to participate All procedures on animals were approved by the Institutional Animal Care and Use Committee at the Faculty of Veterinary Medicine Cairo University with a number: Vet CU18042024892.All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The detection of Leishmania tarentolae in sympatric areas where Leishmania infantum is endemic raised questions regarding the protective effect exerted in dogs by L. tarentolae when in coinfection. This study aimed monitoring the in vitro gene expression of pro- (IFN- γ; TNF-α; IL-12) and anti-inflammatory (IL-4; IL-6; IL-10) cytokines in primary canine macrophages infected by L. tarentolae and L. infantum in single and in co-infections. Macrophages differentiated from dog blood mononuclear cells were infected with the L. tarentolae field-isolated (RI-325) and laboratory (LEM-124) strains, with L. infantum laboratory strain (IPT1), or both. Infection and the number of amastigotes per infected cell were evaluated microscopically by counting a total of 200 cells between 4 and 96 h. Cytokine gene expression was analyzed by real-time PCR from infected macrophages mRNA. Single infections presented higher expression of the cytokines IL-4 and IL-6, and lower of IL-12. Co-infections induced a lower gene expression of IL-4 and IL-6, and a higher gene expression of IL-12, correlating with the low amastigote burden despite the slight increase of infected cells. Data highlight the potential protective effect of L. tarentolae against L. infantum in co-infection by the reduced anti-inflammatory and increased pro-inflammatory cytokines gene expression, opening new perspectives for a canine vaccine development exploiting the non-pathogenic L. tarentolae., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: There is growing evidence that atrial fibrillation (AF) is a risk factor for cognitive impairment (CI) and dementia in the presence or absence of stroke. The purpose of this study was to explore the mechanism of CI caused by AF., Methods: Eighteen male canines were randomly divided into a sham group, a pacing group, and a pacing + GW4869 group. An experimental model of AF was established by rapid atrial pacing (450 beats/min) for 2 weeks, and the sham group received pacemaker implantation without atrial pacing. The GW4869 group received an intravenous GW4869 injection (0.3 mg/kg, once a day) during pacing. All canines were locally injected with Ad-CD63-RFP in epicardial adipose tissue (EAT) to trace the exosomes. Ultracentrifugation was employed to isolate EAT-derived exosomes, followed by RNA sequencing and quantitative real-time PCR (qRT-PCR) to assess RNA in both exosomes and hippocampal tissue. The miRanda database was used to predict the targeting relationships between miRNA and mRNA, which were further validated by luciferase reporter assays. Western blot analysis was conducted to detect exosomal markers (CD63, CD81, TSG101) in EAT exosomes, while immunofluorescence was used to detect Ad-CD63-RFP signals in both EAT and hippocampal tissues, as well as microglial activation marker IBA-1. To further explore the effects of exosomes on microglial cells, in vitro experiments using brain microvascular endothelial cells (bEnd3) and microglial cells (BV2) were conducted. IBA-1 expression and RNA levels in BV2 cells were analyzed by immunofluorescence and qRT-PCR, respectively., Results: After 14 days of pacing of the canine atrium, compared to the sham group, both the pacing and GW4869 groups exhibited an increased number of AF inductions, along with prolonged AF duration. The fluorescence intensity of Ad-CD63-RFP and the microglial activation marker IBA-1 were markedly greater in the hippocampus. RNA sequencing showed that the differentially expressed gene cfa-miR-22e in EAT exosomes was upregulated, and its target gene IL33 was downregulated in the hippocampus. qRT-PCR showed that the levels of cfa-miR-22e were increased in both EAT exosomes and the hippocampus, while the expression of IL-33, a target of cfa-miR-22e, was decreased in the hippocampus. The administration of GW4869 abolished these effects. The in vitro results from bEnd3 and BV2 cell experiments were consistent with the conclusions drawn from the in vivo studies., Conclusion: Our study indicated that the exosomes secreted by EAT in canines with AF can penetrate the BBB and activate microglia in the hippocampus through the cfa-miR-22e/IL33 signalling pathway., (© 2024. The Author(s).)

Doberman Pinschers are known for their increased susceptibility to dilated cardiomyopathy (DCM) relative to other domestic dogs. This makes the Doberman Pinscher a key model for gene-disease investigations. We conducted a genome-wide association study (GWAS) leveraging a database of genetic profiles obtained through collaboration with the Doberman Diversity Project (DDP). We worked in parallel to increase the depth and power of the DDP database. We exchanged direct correspondences with listed breeders and owners to establish health updates for participant dogs. In total, our study included data on 216, 184 single nucleotide polymorphisms (SNPs) in 46 cases and 3226 population control Doberman Pinschers. Using a generalized linear mixed model and saddlepoint approximation to correct for unbalanced group sizes, we identified a cluster of SNPs associated with DCM on chromosome 16., (© 2024. The Author(s).)

Rationale: Peritonitis caused by Pantoea agglomerans is a rare occurrence in patients undergoing peritoneal dialysis. Cases potentially linked to pet dogs are even rarer, and there is limited literature available., Patient Concerns: A patient undergoing peritoneal dialysis presented with symptoms of peritonitis, including abdominal pain and cloudy dialysis fluid., Diagnoses: Microbiological analysis identified P agglomerans as the causative organism., Interventions: The patient was treated with targeted antibiotic therapy and showed a positive response., Outcomes: During a subsequent medical interview, it was revealed that the patient had close contact with their pet dog, raising the possibility that the infection may have been associated with this exposure., Lessons: This case highlights the importance of considering zoonotic transmission as a potential source of infection in peritoneal dialysis patients, particularly when there is close contact with pets. Healthcare providers should educate patients about the potential risks posed by pets and implement preventive strategies to mitigate such risks., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Canine visceral leishmaniasis (CVL) is a parasitic disease caused by the protozoan Leishmania infantum. Neurological infection occurs due to the parasite's ability to cross the blood-brain barrier. It is known that dogs can remain infected with a subclinical infection for life, potentially acting as reservoirs for L. infantum when bitten by sandflies. In this context, the objective of this study was to evaluate the occurrence of Leishmania spp. in the brains of dogs from the metropolitan region of Rio Grande do Sul, Brazil, without a history of neurological disease but residing in an endemic area for L. infantum. A total of 200 samples, from 2022 to 2023, were evaluated using conventional Polymerase Chain Reaction (PCR) with the primers Leishmini-F GGKAGGGGCGTTCTGC and Leishmini-R STATWTTACACCAACCCC, aiming to amplify a product of 120 base pairs for Leishmania spp. To identify the species, a multiplex PCR was used, differentiating L. braziliensis (127 bp), L. amazonensis (100 bp), and L. infantum (60 bp), with the molecular target being the conserved region of the kinetoplast DNA (kDNA) minicircle, specific to Leishmania spp. Of the 200 samples evaluated, 26.5% (53/200) tested positive in the conventional PCR reaction for Leishmania spp., with the PCR multiplex the only species detected was Leishmania infantum. The average age of the positive animals was 5.08 years, with 47.2% being females and 52.8% being males; among these, mixed-breed dogs were the most predominant, representing 43.4% of the total. Clinical signs varied: hepatomegaly in two dogs, pronounced neutrophilic hepatitis in one, splenomegaly in one with lymphoid hyperplasia, and glomerulonephritis and nephritis in two animals. Mild anemia and thrombocytopenia were found in eight, with pale mucous membranes in three, and diffuse alveolar edema in one case. Notable pathological findings included suspected distemper in one animal and lymphoplasmacytic meningitis in another. Histopathological findings revealed alveolar edema and acute renal failure. A third dog exhibited bilateral hydrocephalus and diffuse edema in the brain. Additional changes, such as mild inflammatory infiltrate and slight vacuolar degeneration, were observed in 11.3% of the analyzed brains. There was no clinical suspicion of leishmaniasis in any of the studied cases. Therefore, the detection of L. infantum DNA in the brains of dogs suggests that animals with subclinical infection may play a crucial role in the spread of leishmaniasis, and infection by Leishmania spp. should be considered as a differential diagnosis for neurological disease in endemic areas for the protozoan., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E.coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E.coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E.coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method enables the pathogenicity analysis and vaccine development using genetically modified viruses, and it is expected to allow for faster analysis of newly emerging variants than ever before. Furthermore, its application to other RNA viruses is also expected., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tanaka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Craniofacial anomalies encompassing the orofacial cleft are associated with > 30% of systemic congenital malformations. Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF) is a rare genetic disorder attributed to variants in the actin beta (ACTB) or actin gamma genes that are correlated with a range of craniofacial abnormalities, including cleft lip and/or palate. The underlying pathological mechanism of BWCFF remains elusive, and it is necessary to investigate the etiology of orofacial clefts in patients with BWCFF. In this study, we identified a missense variant (c.1043C > T: p.S348L) in the ACTB gene of a patient with BWCFF and concomitant cleft lip and palate. Furthermore, we performed functional assessments of this variant using various disease models such as the MDCK cell line and Xenopus laevis. These models revealed a compromised capacity of mutated ACTB to localize to the epithelial junction, consequently affecting the behavior of epithelial cells. Additionally, we discovered that the mutated ACTB exhibited an impaired ability to bind PROFILIN1, a critical factor in actin polymerization. This defective ability may contribute to the molecular etiology of aberrant epithelial cell adhesion and migration, resulting in orofacial cleft formation in BWCFF., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Tick control mainly depends on using chemical acaricides that have led to the emergence of resistant tick populations along with environmental hazards. Natural alternatives including essential oils are now widely used to avoid the undesirable effects of chemicals on human, animals and environment. In this study, three commercial oils (myrrh, patchouli, and cypress) and their nanoemulsions (NEs) were tested against Rhipicephalus sanguineus sensu lato unfed adults. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to characterize the nanoemulsions. Four concentrations with three replicates were used in the bioassay of oils and NEs against ticks using an adult immersion test. Furthermore, a toxicity study of the three oils and their NEs using normal fibroblast cells (BJ-1) was performed. TEM revealed particle size ranges from 29 to 211 nm with spherical droplets. The droplet size and polydispersity index were (29.30 nm & 0.367), (211.6 nm & 0.221) and (164.2 nm & 0.237) for myrrh NE, patchouli NE, and cypress NE, respectively. Myrrh and patchouli oils recorded high acaricidal activity followed by cypress oil with LC 50 value 5 days after treatment of 9.01, 12.40, and 15.21%, respectively. The acaricidal efficacy of oil was potentiated in the NEs form which was proved by the reduction in LC 50 values that were recorded 7 days after treatment 4.17, 8.57, and 5.04%, for myrrh, patchouli, and cypress NE, respectively. As an alternative for lab animals, we applied the cytotoxicity of myrrh, patchouli, and cypress on normal fibroblast cells. The oils showed low cytotoxic activity of 10.6, 21.5, and 23.6% for Myrrh, Patchouli, and Cypress, respectively. The nanoemulsion was revealed to be safe for normal cells up to a concentration of 0.62% (cytotoxicity%= 24.4, 34.4, and 16.3%, respectively), and the moderate cytotoxic effect appeared at around a concentration of 1.25% (cytotoxicity%= 42.2%, 57.0%, and 36.8% respectively). In conclusion, the three oils and their NEs have good acaricidal activity against Rhipicephalus sanguineus unfed adults. Further toxicity studies on mammals are needed to ensure the safe use of these formulations for the control of ticks on infested animals., (© 2024. The Author(s).)

The effort to eradicate Dracunculus medinensis, the etiologic agent of dracunculiasis, or Guinea worm disease, began at CDC in 1980. In 1986, with an estimated 3.5 million global cases in 20 African and Asian countries, the World Health Assembly called for dracunculiasis elimination. The Guinea Worm Eradication Program (GWEP) was established to help countries with endemic dracunculiasis reach this goal. GWEP is led by The Carter Center and supported by partners, including the countries with endemic disease, CDC, UNICEF, and the World Health Organization. Since 2012, infections in dogs, cats, and baboons have posed a new challenge for GWEP, as have ongoing civil unrest and insecurity in some areas. As of June 2024, dracunculiasis remained endemic in five countries (Angola, Chad, Ethiopia, Mali, and South Sudan). Fourteen human cases and 886 animal infections occurred, including 407 dogs in Chad and 248 dogs in Cameroon, reported in 2023, and three human cases and 297 animal infections reported during January-June 2024. Animal infections, primarily in dogs in Cameroon and Chad, and impeded access due to civil unrest and insecurity in Mali, threaten the near-term possibility of global eradication. Nevertheless, countries appear poised to reach zero cases., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Introduction: Rabies virus infection can cause fatal brain disease in mammals. Any species is susceptible to infection. Any effort aimed at recognizing infected animals and performing first actions in the event of transmission is mandatory., Objective: To determine knowledge, attitudes, and practice profiles regarding rabies, or hydrophobia, in a municipality of Colombia, using a multiple correspondence analysis., Materials and Methods: A descriptive observational study involving 71 pet owners was carried out in the municipality of Ibagué (Tolima). A questionnaire-based survey collected data on rabies knowledge, attitudes, practices, and demographic information. The survey was conducted between October and November 2021, and the data analysis involved descriptive statistics and multiple correspondence analysis., Results: The study revealed a commendable level of rabies awareness among the urban residents in the study municipality. However, there are causes of concern as they allow stray animals to enter their homes and are not aware of the importance of notifying dead animals. The respondents demonstrated a humane approach to bite management and emphasized the importance of wound cleaning. Additionally, they expressed a strong desire for more information to enhance their knowledge and awareness of the disease., Conclusion: The findings of this study provide valuable insights for improving rabies prevention efforts and promoting public health. Health education, evidence-based strategies, and community participation are essential for successful disease control and educational gaps addressing related to sociocultural factors.

Background: Iron-deficiency is associated with increased morbidity and mortality in non-communicable diseases. However, iron parameters are rarely assessed in dogs. Here, we aimed to assess and correlate iron parameters in dogs suffering from Canine Atopic Dermatitis (CAD) compared to non-atopic, healthy dogs., Results: For this retrospective study, blood values and sera of 34 dogs with confirmed CAD were compared with 94 healthy non-atopic dogs. In our cohort, dogs with CAD had significantly lower mean corpuscular volume (MCV, ) mean corpuscular hemoglobin (MCH) but higher white blood cell counts due to increased levels of circulating neutrophils and monocytes. CAD patients also had elevated total protein and c-reactive protein (CRP), but lower albumin levels compared to our healthy control dogs, indicated low-grade inflammation in the CAD cohort. Spearman correlations associated negatively clinical symptom (CADESI-4/PVAS) with MCV; ceruloplasmin and hepcidin, but positively with serum iron. Only in the CAD-cohort, MCV, CRP and albumin-levels negatively affected serum iron-levels and were positively associated with ceruloplasmin. Linear regression analysis revealed that serum iron-levels in CAD subjects, were positively dependent on hematocrit (packed cell volume, PCV) and albumin, and negatively dependent with white blood cells and neutrophils numbers. In contrast, in the healthy cohort, hepcidin was the sole factor associated with serum iron., Conclusions: A decreased iron status was associated with a higher symptom burden. Iron homeostasis differed markedly in healthy and atopic dermatitis dogs. CAD patients had depleted iron-stores and presented themselves with subclinical inflammation., (© 2024. The Author(s).)

The rapid advancements in sequencing technologies and bioinformatics have enabled metagenomic research of complex microbial systems, but reliable results depend on consistent laboratory and bioinformatics approaches. Current efforts to identify best practices often focus on optimizing specific steps, making it challenging to understand the influence of each stage on microbial population analysis and compare data across studies. This study evaluated DNA extraction, library construction methodologies, sequencing platforms, and computational approaches using a dog stool sample, two synthetic microbial community mixtures, and various sequencing data sources. Our work, the most comprehensive evaluation of metagenomic methods to date. We developed a software tool, termed minitax, which provides consistent results across the range of platforms and methodologies. Our findings showed that the Zymo Research Quick-DNA HMW MagBead Kit, Illumina DNA Prep library preparation method, and the minitax bioinformatics tool were the most effective for high-quality microbial diversity analysis. However, the effectiveness of pipelines or method combinations is sample-specific, making it difficult to identify a universally optimal approach. Therefore, employing multiple approaches is crucial for obtaining reliable outcomes in microbial systems., (© 2024. The Author(s).)

Purpose: When performing thoracic aortic endovascular repair (TEVAR) on lesions of the aortic arch, physician-modified fenestration or in situ fenestration is often used to maintain patent branches. We designed a new adjustable prefenestration aortic stent graft that can both isolate pathologies in the aortic arch and obtain patent branches simultaneously. In this study, we use this new type of stent to perform fenestrated TEVAR in a canine’s aorta. This study aims to evaluate the safety and feasibility of the new device, which may provide preliminary data for potential human application., Methods: Eight Labrador Retriever canines underwent fenestrated TEVAR using the new stent device. Digital subtract angiography (DSA) was performed before and after fenestrated TEVAR to evaluate the safety and feasibility of the procedure. For the device deployment, at the “large curvature” side in the endograft, there is a rectangular prefenestration area (2 × 5 cm) without the polytetrafluoroethylene membrane, and at both longer side edges of the fenestration, there are two slide rails. A moveable membrane that covers the same area as the prefenestration area is initially set at the prefenestration position. A stay line is connected from the distal site of the moveable membrane that controls it to the distal position along the slide rail, which releases the fenestration. After the positioning of the prefenestration is determined, the outer sheath of the delivery system is released, and the stay line at the end of the delivery system is pulled outside the body. The animals were divided into a 1-month group (n = 4) and a 3-month group (n = 4) after the fenestrated TEVAR. Computed tomography (CT) was performed before euthanasia, and video of the DSA during the procedures and CT angiography (CTA) images were then studied., Results: The procedure success rate was 100%, but the total survival rate was only 87.5%. There were no aortic-related deaths during follow-up, and during the operation, there were no stent-graft-related accidents. In addition, no stent-graft migrations were observed in the CTA, and all branch arteries were kept patent by the adjustable fenestration. Finally, histological examination and electron microscope results showed no obvious vascular injury or inflammation., Conclusion: Based on the results of this study, we judge the safety and feasibility of the use of the newly designed adjustable prefenestration aortic stent graft in a fenestrated-TEVAR canine model to be acceptable. Our preliminary data may serve as an initial reference for evaluating the potential application of the new stent in humans., Competing Interests: Conflict of interest disclosure: The authors declared no conflicts of interest.

Purpose: Synthesis and characterization of silver (Ag)/titanium dioxide (TiO 2 ) nanocomposite (ATA) to investigate its antiviral activity against the H1N1 influenza virus and antiviral mechanisms., Materials and Methods: A water-dispersible ATA was prepared by a photocatalytic reduction process from AgNO 3 and TiO 2 . The characterization of ATA was performed by ultraviolet-visible spectroscopy, X-ray diffraction, high-resolution transmission electron microscopy and energy-dispersive X-ray spectroscopy. The antiviral activities and the antiviral mechanism of ATA were investigated in detail by light microscopy, transmission electron microscopy and biological techniques such as cell cytotoxicity, 50% tissue culture infectious dose detection, western blot and reverse transcription-polymerase chain reaction., Results: These results showed the successful synthesis of ATA nanocomposite with uniform particle size and distribution. It demonstrated the highly efficient antiviral activity of ATA in a dose- and time-dependent manner, as indicated by the reduction of viral titer and the reduction of cytopathic effects caused by viral infection. In the presence of ATA, the structure of the H1N1 influenza virus is directly destroyed and even disintegrated, with the damaged surface membrane proteins and fuzzy contour. It reduces the infection efficiency of influenza by suppressing the activity and expression of hemagglutinin and neuraminidase. The results of mechanistic studies suggested that ATA nanocomposite primarily interferes with virus attachment to viral receptors on the cell surface., Conclusion: Our study suggests that ATA may be a good antiviral candidate against the influenza virus. Compared with AgNPs alone, our synthesized ATA nanocomposites can achieve similar viral inactivation rates using only a much smaller concentration of AgNPs, greatly reducing the amount of AgNPs and their potential side effects. It has great practical value for attaching ATA to the high-efficiency particulate air network in the air purifier, which can kill the virus attached to it and limit its spread., Competing Interests: The author(s) report no conflicts of interest in this work., (© 2024 Ma et al.)

The sleep spindle-generating thalamo-cortical circuitry supports attention capacity in awake humans and animals, but using sleep spindles to predict differences in attention has not been tried in either. Of the more commonly examined cognitive correlates of spindle occurrence and amplitude, post-sleep recall, and general intelligence, only post-sleep recall had been studied in dogs, rats and mice. Here, we examined a sample of companion dogs (N = 58) for whom polysomnographic recordings and several cognitive tests were performed on two occasions each, with a three-month break in-between. Five of the tests were used to extract a factor analogous to human g (general mental ability). A sixth test in the battery measured sustained attention. Both attention and g-factor scores were linked to higher slow spindle occurrence and absolute sigma power detected in polysomnographic recordings over the central electrode. These effects persisted across measurement occasions. Higher intrinsic spindle frequency was, in turn, linked to lower g-factor scores but displayed no relationship with attention scores. The overlap in localization and direction for the effects of slow spindle density (spindles/minute) and sigma power supports that they tap into the same underlying cognition-relevant aspects of spindling. Given earlier large sample and meta-analysis validations of sigma power as a reliable predictor of cognitive performance in humans, we thus conclude that the currently handled method for quantifying spindle density in dogs indeed measures cognition-relevant spindle activity by virtue of its agreement with the sigma power alternative., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Canine parvovirus (CPV) can cause high morbidity and mortality rates in puppies, posing a significant threat to both pet dogs and the breeding industry. Rapid, accurate, and convenient detection methods are important for the early intervention and treatment of canine parvovirus. In this study, we propose a visual CPV detection system called nucleic acid mismatch enzyme digestion (NMED). This system combines loop-mediated isothermal amplification (LAMP), endonuclease for gene mismatch detection, and colloidal gold lateral chromatography. We demonstrated that NMED can induce the binding of the amplicon from the sample to the specific labeling probe, which in turn triggers digestion by the endonuclease. The sensitivity and visual visibility of LAMP were increased by combining endonuclease and colloidal gold lateral chromatography assisted by a simple temperature-controlled device. The sensitivity of the NMED assay was 1 copy/μL, which was consistent with quantitative PCR (qPCR). The method was validated with 20 clinical samples that potentially had CPV infection; 15 positive samples and 5 negative samples were evaluated; and the detection accuracy was consistent with that of qPCR. As a rapid, accurate, and convenient molecular diagnostic method, NMED has great potential for application in the field of pathogenic microorganism detection., Importance: The NMED method has been established in the laboratory and used for CPV detection. The method has several advantages, including simple sampling, high sensitivity, intuitive results, and no requirement for expensive equipment. The establishment of this method has commercial potential and offers a novel approach and concept for the future development of clinical detection of pathogenic microorganisms., Competing Interests: The authors declare no conflict of interest.