Seroepidemiological survey to cell culture rabies vaccines (CCRV) in Brazil.

Rabies is a contagious viral disease that can be easily transmitted by the saliva and brain/nervous system tissues of the infected animals, causing severe and fatal encephalitis in both animals and humans. Vaccination campaigns are crucial to combat and prevent rabies's spread in dogs and humans. The Modified Fuenzalida & Palicios vaccines have been widely used since the 70s and have proven effective in producing a solid serological response. Since 2008, the Brazilian Ministry of Health has introduced a Cell Culture Rabies Vaccine (CCRV) for all dog mass vaccination campaigns in Brazil. However, to date, there is limited evidence on the immunologic response of dogs to this type of vaccine in field conditions. The present study evaluated the serological response in dogs vaccinated with CCRV from blood samples of 724 dogs using the Simplified Fluorescence Inhibition Microtest - SFIMT. Dogs with a titer equal to 0.5 IU/mL or above were considered seropositive. The results revealed that 59.12% (428/724) of all dogs tested and 48.49% (32/66) of primo-vaccinated animals were seropositive. The percentage of seronegative animals was higher than seropositive for animals that received a single dose during their life (p < 0.05). The opposite was observed in animals with five or more doses. The results of this study demonstrated that the CCRV vaccines elicit a satisfactory immunological response in field conditions and can constitute an essential population-level preventive strategy as part of annual canine rabies vaccination campaigns. Although its effectiveness has been studied, there is limited evidence of its immunological response in dogs under field conditions. This paper evaluates the serological response to CCRV in dogs vaccinated during mass vaccination campaigns from 2012 to 2017.

Human assembloid model of the ascending neural sensory pathway.

The ascending somatosensory pathways convey crucial information about pain, touch, itch, and body part movement from peripheral organs to the central nervous system. Despite a significant need for effective therapeutics modulating pain and other somatosensory modalities, clinical translation remains challenging, which is likely related to species-specific features and the lack of in vitro models to directly probe and manipulate this polysynaptic pathway. Here, we established human ascending somatosensory assembloids (hASA)- a four-part assembloid completely generated from human pluripotent stem cells that integrates somatosensory, spinal, diencephalic, and cortical organoids to model the human ascending spinothalamic pathway. Transcriptomic profiling confirmed the presence of key cell types in this circuit. Rabies tracing and calcium imaging showed that sensory neurons connected with dorsal spinal cord projection neurons, which ascending axons further connected to thalamic neurons. Following noxious chemical stimulation, single neuron calcium imaging of intact hASA demonstrated coordinated response, while four-part concomitant extracellular recordings and calcium imaging revealed synchronized activity across the assembloid. Loss of the sodium channel SCN9A, which causes pain insensitivity in humans, disrupted synchrony across the four-part hASA. Taken together, these experiments demonstrate the ability to functionally assemble the essential components of the human sensory pathway. These findings could both accelerate our understanding of human sensory circuits and facilitate therapeutic development.

Circuit mechanism underlying fragmented sleep and memory deficits in 16p11.2 deletion mouse model of autism.

Sleep disturbances are prevalent in children with autism spectrum disorder (ASD) and have a major impact on the quality of life. Strikingly, sleep problems are positively correlated with the severity of ASD symptoms, such as memory impairment. However, the neural mechanisms underlying sleep disturbances and cognitive deficits in ASD are largely unexplored. Here, we show that non-rapid eye movement sleep (NREMs) is highly fragmented in the 16p11.2 deletion mouse model of ASD. The degree of sleep fragmentation is reflected in an increased number of calcium transients in the activity of locus coeruleus noradrenergic (LC-NE) neurons during NREMs. Exposure to a novel environment further exacerbates sleep disturbances in 16p11.2 deletion mice by fragmenting NREMs and decreasing rapid eye movement sleep (REMs). In contrast, optogenetic inhibition of LC-NE neurons and pharmacological blockade of noradrenergic transmission using clonidine reverse sleep fragmentation. Furthermore, inhibiting LC-NE neurons restores memory. Rabies-mediated unbiased screening of presynaptic neurons reveals altered connectivity of LC-NE neurons with sleep- and memory regulatory brain regions in 16p11.2 deletion mice. Our findings demonstrate that heightened activity of LC-NE neurons and altered brain-wide connectivity underlies sleep fragmentation in 16p11.2 deletion mice and identify a crucial role of the LC-NE system in regulating sleep stability and memory in ASD.

Detection of ectoparasites and investigation of infection by Rickettsia spp. and Trypanosoma spp. in bats from Rio Grande do Sul, Brazil.

This study aimed to investigate the presence of ectoparasites and the occurrence of natural infection by Rickettsia spp. and Trypanosoma spp. in bats from Rio Grande do Sul (RS), Brazil. The evaluated animals were obtained from the Instituto de Pesquisas Veterinárias Desidério Finamor, sent by the Centro Estadual de Vigilância Sanitária, to carry out rabies diagnostic tests, during the period from 2016 to 2021. The bats came from 34 municipalities in RS. Of the 109 animals surveyed, 35.8% (39/109) had 385 ectoparasites, with an average of 9.9 parasites per animal. Of these bats, all had insectivorous feeding habits, with 35.9% (14/39) females and 64.1% (25/39) males. The co-parasitism of Chirnyssoides sp., Ewingana inaequalis, and Chiroptonyssus robustipes on Molossus currentium (Mammalia, Chiroptera) was recorded for the first time. All bats surveyed were negative for infection by the protozoan and bacteria. Thus, the expansion of the occurrence of these ectoparasites in insectivorous bats in RS was observed. Furthermore, this study corresponds to the first recorded interspecific associations for the species.

Biomimetic nanocarriers loaded with temozolomide by cloaking brain-targeting peptides for targeting drug delivery system to promote anticancer effects in glioblastoma cells.

Glioma is the leading cancer of the central nervous system (CNS). The efficacy of glioma treatment is significantly hindered by the presence of the blood-brain barrier (BBB) and blood-brain tumour barrier (BBTB), which prevent most drugs from entering the brain and tumours. Hence, we established a novel drug delivery nanosystem of brain tumour-targeting that could self-assemble the method using an amphiphilic Zein protein isolated from corn. Zein's amphiphilicity prompted it to self-assembled into NPs, efficiently containing TMZ. This allowed us to investigate temozolomide (TMZ) for glioblastoma (GBM) treatment. To construct TMZ-encapsulated NPs (TMZ@RVG-Zein NPs), the NPs' Zein was clocked to rabies virus glycoprotein 29 (RVG29). To verify that the NPs could penetrate the BBB and precisely target and kill the GBM cancer cell line, in vitro studies were performed. The process of NPs penetrating cancer cell membranes was investigated using enzyme-linked immunosorbent assays (ELISAs) to measure the expressions of nicotinic acetylcholine receptors (nAChRs) on the U87 cell line. Therefore, effective targeted brain cancer treatment is possible by forming NP clocks, a cell-penetrating natural Zein protein with an RVG29. These NPs can penetrate the blood-brain barrier (BBB) and enter the glioblastoma (U87) cell line to release TMZ. These NPs have a distinct cocktail of biocompatibility and brain-targeting abilities, making them ideal for involving brain diseases.

The matrix protein of lyssavirus hijacks autophagosome for efficient egress by recruiting NEDD4 through its PPxY motif.

Lyssaviruses are well-known worldwide and often cause fatal encephalitis. Previous studies have shown that autophagy is beneficial for the replication of rabies virus (RABV), the representative lyssavirus, but the detailed mechanism remains obscure. In this study, we showed that the rabies virus matrix protein (RABV-M) used its PPxY motif to interact with the E3 ubiquitin-protein ligase NEDD4. NEDD4 then recruited MAP1LC3/LC3 via its LC3-interacting region (LIR). Interestingly, after binding to the ubiquitinated RABV-M, NEDD4 could bind more LC3 and enhance autophagosome accumulation, while NEDD4 knockdown significantly reduced M-induced autophagosome accumulation. Further study revealed that RABV-M prevented autophagosome-lysosome fusion and facilitated viral budding. Inhibition of RABV-M-induced autophagosome accumulation reduced the production of extracellular virus-like particles. We also found that M proteins of most lyssaviruses share the same mechanism to accumulate autophagosome by hijacking NEDD4. Collectively, this study revealed a novel strategy for lyssaviruses to achieve efficient viral replication by exploiting the host autophagy system.Abbreviations: ABLV: Australian bat lyssavirus; ATG5: autophagy related 5; Baf A1:bafilomycin A1;co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI:4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EBLV:European bat lyssavirus; GFP: green fluorescent protein; GST:glutathione S-transferase; hpi: hours post-infection; hpt: hourspost-transfection; LIR: LC3-interactingregion;MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry:red fluorescent protein; MOI: multiplicity of infection; NC: negativecontrol; MVB: multivesicular body; NEDD4: neural precursorcell-expressed developmentally down-regulated 4; RABV: rabies virus;SQSTM1/p62: sequestosome 1; VLP: virus-like particle; VPS4B: vacuolarprotein sorting 4B; TEM: transmission electron microscopy; WB:western blotting; WT: wild-type; µm: micrometer; µM: micromole.

Comparative Study on the Immunogenicity and Efficacy of Different Post-exposure Intramuscular Rabies Vaccination Regimens in China.

Objective: This study aimed to compare the current Essen rabies post-exposure immunization schedule (0-3-7-14-28) in China and the simple 4-dose schedule (0-3-7-14) newly recommended by the World Health Organization in terms of their safety, efficacy, and protection. Methods: Mice were vaccinated according to different immunization schedules, and blood was collected for detection of rabies virus neutralizing antibodies (RVNAs) on days 14, 21, 28, 35, and 120 after the first immunization. Additionally, different groups of mice were injected with lethal doses of the CVS-11 virus on day 0, subjected to different rabies immunization schedules, and assessed for morbidity and death status. In a clinical trial, 185 rabies-exposed individuals were selected for post-exposure vaccination according to the Essen schedule, and blood was collected for RVNAs detection on days 28 and 42 after the first immunization. Results: A statistically significant difference in RVNAs between mice in the Essen and 0-3-7-14 schedule groups was observed on the 35th day ( P < 0.05). The groups 0-3-7-14, 0-3-7-21, and 0-3-7-28 showed no statistically significant difference ( P > 0.05) in RVNAs levels at any time point. The post-exposure immune protective test showed that the survival rate of mice in the control group was 20%, whereas that in the immunization groups was 40%. In the clinical trial, the RVNAs positive conversion rates on days 28 (14 days after 4 doses) and 42 (14 days after 5 doses) were both 100%, and no significant difference in RVNAs levels was observed ( P > 0.05). Conclusion: The simple 4-dose schedule can produce sufficient RVNAs levels, with no significant effect of a delayed fourth vaccine dose (14-28 d) on the immunization potential.

In Commemoration of Dr. Mostafa Pourtaghva Shahrestani, a Pioneer in Infectious Disease Research.

It is important to honor the contributions of scientific leaders who have dedicated their lives to advancing knowledge and serving their country. One way is to document their experiences and personalities in a documentary format, which can serve as a historical record and an inspiration for future generations. Dr. Mostafa Pourtaghva Shahrestani, a renowned physician and specialist in infectious diseases and tropical medicine, has made significant contributions to public health in Iran. He has played a crucial role in controlling infectious diseases such as smallpox, tuberculosis, rabies, plague, and cholera. Throughout his career, he has held various executive positions, including the head of Pasteur Hospital and the director of the Pasteur Institute of Iran. Dr. Pourtaghva's life is a testament to his unwavering dedication to public health services, as evidenced by his continuous effort, love, and interest in honest work. His inspiring story can serve as a model for those who seek to follow in his footsteps.

CASE REPORT: Fulminant acute hemorrhagic Leukoencephalitis (AHLE): A rare and ruinous outcome with cerebral herniation (COVID-19).

Background: Acute hemorrhagic leukoencephalitis (AHLE) is a very rare demyelinating disease with rapid fulminant inflammation of the white matter. Although the exact etiology is unknown, AHLE usually manifests post a viral or bacterial infection and less often seen post vaccination for measles or rabies. AHLE has a very poor prognosis and a high mortality rate. Owing to the rarity of this entity there is not clear consensus on the proper line of management. In this report, we present a case of AHLE as a para-infectious sequel to COVID-19 in a young patient. Clinical presentation: We report a 30-year-old turkish patient with an initial presentation of upper respiratory tract infection due to COVID-19. Initially, she was admitted to the hospital with generalized tonic-clonic seizure (GTCS) and deterioration in her level of consciousness lapsing into a coma. An initial CT scan showed diffuse brain edema and an MRI head confirmed the suspicion of Acute hemorrhagic leukoencephalitis (AHLE). Despite prompt and diligent osmotic therapy and pulsed intravenous (IV) methylprednisolone, her condition rapidly depreciated and progressed into cerebral edema with gravid sequela of brainstem herniation. Conclusions: AHLE is a very rare entity and perhaps its fulminant debilitating course and high mortality should warrant further studies on disease pathophysiology and its optimal treatment parameters. Life-saving decompressive hemicraniectomy should be considered in the multidisciplinary approach of the management with tailored osmotic and immunotherapy.

Comparison of 4 laboratory tests for the detection of bovine rabies viral infection in Paraguay: fluorescent antibody test, rapid detection test, histologic lesions, and RT-PCR.

Rabies virus (RABV; Lyssavirus rabies) is a neurotropic virus that can be transmitted to mammals by the hematophagous bat Desmodus rotundus. An accurate, accessible method for the detection of RABV in cattle is necessary in Paraguay; thus, we evaluated the detection of RABV using 4 techniques: fluorescent antibody test (FAT), immunochromatography rapid detection test (RDT; Anigen Rapid Rabies Ag test kit; Bionote), a reverse-transcription PCR (RT-PCR) assay, and histologic lesions in different portions of the CNS of 49 Paraguayan cattle to determine the most sensitive and specific technique. By FAT and RDT, 15 of 49 (31%) samples were positive. By RT-PCR amplification of N and G genes, 13 of 49 (27%) and 12 of 49 (25%) were positive, respectively. RDT had high agreement with FAT (kappa = 1); sensitivity was 100% (95% CI: 97-100%) and specificity was 100% (95% CI: 99-100%). The amplification of the N and G genes resulted in substantial agreement (kappa of 0.9 and 0.8, respectively) compared with FAT, and the sensitivity and specificity of the N gene were 87% (95% CI: 66-100%) and 100% (95% CI: 98-100%), respectively, and those of the G gene were 80% (95% CI: 56-100%) and 100% (95% CI: 98-100%), respectively. Histologic lesions observed were lymphoplasmacytic meningoencephalitis, gliosis, and neuronophagia. The agreement observed between the FAT and RDT tests suggests that RDT is an accurate tool for the detection of RABV. Histopathology can be used to confirm lesions caused by RABV and to rule out other conditions; the RT-PCR assay is useful for molecular epidemiology studies.

Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged <2 years old is efficient to stop rabies circulation in Cambodia.

Rabies control remains challenging in low and middle-income countries, mostly due to lack of financial resources, rapid turnover of dog populations and poor accessibility to dogs. Rabies is endemic in Cambodia, where no national rabies vaccination program is implemented. The objective of this study was to assess the short and long-term vaccination-induced immunity in Cambodian dogs under field conditions, and to propose optimized vaccination strategies. A cohort of 351 dogs was followed at regular time points following primary vaccination only (PV) or PV plus single booster (BV). Fluorescent antibody virus neutralization test (FAVNT) was implemented to determine the neutralizing antibody titer against rabies and an individual titer ≥0·5 IU/mL indicated protection. Bayesian modeling was used to evaluate the individual duration of protection against rabies and the efficacy of two different vaccination strategies. Overall, 61% of dogs had a protective immunity one year after PV. In dogs receiving a BV, this protective immunity remained for up to one year after the BV in 95% of dogs. According to the best Bayesian model, a PV conferred a protective immunity in 82% of dogs (95% CI: 75-91%) for a mean duration of 4.7 years, and BV induced a lifelong protective immunity. Annual PV of dogs less than one year old and systematic BV solely of dogs vaccinated the year before would allow to achieve the 70% World Health Organization recommended threshold to control rabies circulation in a dog population in three to five years of implementation depending on dog population dynamics. This vaccination strategy would save up to about a third of vaccine doses, reducing cost and time efforts of mass dog vaccination campaigns. These results can contribute to optimize rabies control measures in Cambodia moving towards the global goal of ending human death from dog-mediated rabies by 2030.

Prioritization of zoonoses of wildlife origin for multisectoral one health collaboration in Guyana, 2022.

Background: The human population in Guyana, located on the South American continent, is vulnerable to zoonotic diseases due to an appreciable reliance on Neotropical wildlife as a food source and for trade. An existing suboptimal health surveillance system may affect the effective monitoring of important zoonotic diseases. To effectively address this deficit, a One Health zoonotic disease prioritization workshop was conducted to identify nationally significant zoonoses. Methods: Prioritization of zoonotic diseases was conducted for the first time in Guyana & Caribbean region using literature review, prioritization criteria and a risk prioritization tool in combination with a consultative One Health workshop. This involved multisectoral experts from varied disciplines of social, human, animal, and environmental health to prioritize zoonotic diseases using a modified semi-quantitative One Health Zoonotic Disease Prioritization (OHZDP) tool. The inclusion and exclusion criteria were applied to pathogen hazards in existence among wildlife in Guyana during the hazard identification phase. Results: In total, fifty zoonoses were chosen for prioritization. Based on their weighted score, prioritized diseases were ranked in order of relative importance using a one-to-five selection scale. In Guyana, this zoonotic disease prioritization method is the first significant step toward bringing together specialists from the fields of human, animal, and environmental health. Following discussion of the OHZDP Tool output among disease experts, a final zoonotic disease list, including tuberculosis, leptospirosis, gastroenteritis, rabies, coronavirus, orthopoxvirus, viral hemorrhagic fevers, and hepatitis were identified as the top eight priority zoonoses in Guyana. Conclusions: This represents the first prioritization of nationally significant zoonotic diseases in Guyana and the English-speaking Caribbean. This One Health strategy to prioritize these eight zoonoses of wildlife origin is a step that will support future tracking and monitoring for disease prevalence among humans and wildlife and can be used as a decision-making guide for policymakers and stakeholders in Guyana.

Magnetic resonance imaging in rabies encephalitis, a case report, and review of the literature.

Rabies is an acute fatal disease of the central nervous system. Neuroimaging plays an important role, especially in establishing an early diagnosis and distinguishing it from other types of encephalitis. This case report aims to give a brief review of this condition and report the less common MRI findings of the disease. We herein report a case of a 61-year-old male bitten by a stray dog who presented with fever, vomiting, headache, sialorrhea, dysarthria, dysphagia, and upper limb weakness which progressed to lower limbs on the next day. T2W and FLAIR images demonstrated subtle bilateral hyperintense signal in the deep gray matter with more apparent increased signal intensity in the white matter of the frontal and parietal lobes which shows mild diffusion restriction but no postcontrast enhancement. The diagnosis of rabies encephalitis was made based on a typical history of exposure, a compatible clinical presentation, and MRI findings. Rabies diagnosis is essentially clinical. It is definitively confirmed by the isolation of the virus from biological samples such as saliva, CSF, hair, or detection of rabies antigens or antibodies. Magnetic resonance imaging (MRI) brain used as one of the modalities of investigation for distinguishing it from other encephalitis. Rabies per se does not have any characteristic features on the MRI brain.

Burden of dog bite injuries and wound management practices to prevent rabies among dog owners.

Background: Despite members of dog-owning families being at a higher risk of dog bites owing to their proximity to dogs in their household, there are hardly any studies from India which focus on the burden of dog bites among them and their rabies control and prevention practices. This study aimed to estimate the burden of dog bites among dog owners and their wound management practices to prevent rabies. Materials and Methods: A cross-sectional study was conducted among pet dog owners in a high-end housing society of National Capital Region of Delhi. A pre-tested and pre-validated schedule was used to collect data by consecutive sampling through community survey. Data were analyzed using R software. The incidence of dog bites and the status of rabies prevention practices adopted by the participants are presented as proportions. Chi-square test was applied to compare proportions. Results: A total of 100 families were studied, which covered 355 family members. The incidence of dog bites in the past 1 year was found to be 44/355, 12.4% (95% confidence interval 9.2-16.3%). Pet dogs were responsible for 31/44 (70.5%) bites. Among 44 dog bite incidents, 30 (68.2%) reported taking any injection after the incident, and 10 (22.7%) reported receiving an anti-rabies vaccine. Only six out of 100 families reported at least one family member covered by rabies pre-exposure prophylaxis. Conclusion: The incidence of dog bites among the dog owners was high. The rabies pre- and post-exposure prophylaxis practices adopted by the participants were found to be inadequate.

Risk of rabies reintroduction into the European Union as a result of the Russo-Ukrainian war: A quantitative disease risk analysis.

AIMS: The importation of rabid animals poses a continual threat to rabies freedom in the European Union (EU). Following the Russian invasion of Ukraine in 2022, the European Commission eased the rules on companion animal importations for Ukrainian refugees through derogations to the EU Pet Travel Scheme (EU PETS). As Ukraine remains endemic for canine rabies, this paper aimed to quantitatively assess whether the change in regulations affected the risk of rabies introduction to the EU. METHOD AND RESULTS: Transmission pathways for both EU PETS and derogations to this scheme were considered, as well as the scenarios of 100% compliance and reduced compliance within both schemes. Stochastic modelling was performed via a Monte Carlo simulation to calculate the median and 95% confidence intervals of the annual risk of rabies entry and the years between rabies entries into the EU. Following 100% compliance, the derogation scheme posed a significantly lower risk at a value of 3.63 × 10-3 (CI 95% 1.18 × 10-3-9.34 × 10-3) rabies entries per year in comparison to EU PETS where the risk was 4.25 × 10-2 (CI 95% 1.44 × 10-2-9.81 × 10-2). Despite a significantly lower risk as compared to EU PETS under 100% compliance, the derogation scheme was more sensitive to a reduction in compliance which resulted in a 74-fold increase in risk. Given this, even under reduced compliance, the annual risk remained lower under the derogation scheme, although this was not statistically significant. CONCLUSION: The findings of this study suggest that a 4-month period of quarantine, as modelled for the derogation scenario, could reduce the annual risk of rabies entry. This scheme may present a viable solution for the management of companion animal influxes in future crises.

A tale of endurance: bats, viruses and immune dynamics.

The emergence of highly zoonotic viral infections has propelled bat research forward. The viral outbreaks including Hendra virus, Nipah virus, Marburg virus, Ebola virus, Rabies virus, Middle East respiratory syndrome coronavirus, SARS-CoV and the latest SARS-CoV-2 have been epidemiologically linked to various bat species. Bats possess unique immunological characteristics that allow them to serve as a potential viral reservoir. Bats are also known to protect themselves against viruses and maintain their immunity. Therefore, there is a need for in-depth understanding into bat-virus biology to unravel the major factors contributing to the coexistence and spread of viruses.

Bats are the most diverse mammalian order, with over 1400 species found worldwide. Studies on bats have revealed that they frequently carry and transmit multiple viruses. They are also known to recover from viral infections. Further, human interference and climatic changes in bats' native habitat have led to virus spillover events from bats to human populations, posing a serious public health risk. A deeper understanding of the coexistence of bats and viruses, as well as the mechanisms of disease transmission to humans, is required to minimize the risk of future viral outbreaks.

Protective Activity of Inactivated Rabies Vaccine Using Flagellin-Based Adjuvant.

Rabies is a zoonotic disease with high lethality. Most human deaths are associated with the bites received from dogs and cats. Vaccination is the most effective method of preventing rabies disease in both animals and humans. In this study, the ability of an adjuvant based on recombinant Salmonella typhimurium flagellin to increase protective activity of the inactivated rabies vaccine in mice was evaluated. A series of inactivated dry culture vaccine for dogs and cats "Rabikan" (strain Shchelkovo-51) with addition of an adjuvant at various dilutions were used. The control preparation was a similar series of inactivated dry culture vaccine without an adjuvant. Protective activity of the vaccine preparations was evaluated by the NIH potency test, which is the most widely used and internationally recommended method for testing effectiveness of the inactivated rabies vaccines. The value of specific activity of the tested rabies vaccine when co-administered with the adjuvant was significantly higher (48.69 IU/ml) than that of the vaccine without the adjuvant (3.75 IU/ml). Thus, recombinant flagellin could be considered as an effective adjuvant in the composition of future vaccine preparations against rabies virus.

Factors associated with adherence to the principles of responsible companion animal guardianship in a municipality in southeastern Brazil.

Responsible companion animal guardianship (RCAG) covers aspects that are relevant to both animal and human health. Understanding the factors associated with adherence to RCAG principles can guide accountability, animal welfare and disease control. The present observational study describes the level of adherence to RCAG by guardians living in a medium-sized Brazilian municipality and identifies the factors associated with inadequate guardianship. Questionnaires were administered to randomly selected guardians of 704 dogs. The binary outcome of the study, namely more and less adequate compliers to RCAG principles, was analyzed using a score list comprising eight variables relating to the care provided to the dogs, namely provision of veterinary services, vaccination against rabies, deworming, no access to the streets without supervision, walking with guardian, dog freedom at home, registration (microchipping) and satisfactory food supply. Factors possibly associated with less adequate adherence to RCAG by the guardian, such as socioeconomic and cultural features of the guardians, characteristics of the dogs and attributes of the guardian-animal interactions, were analyzed using multiple logistic regression models. The RCAG actions least adopted by guardians were animal registration, provision of veterinary care, walking with the dog and preventing access of the dogs to the streets without supervision. Individuals who cared for a single dog, a mixed breed dog or had previously lost a dog were less likely to show adequate adherence to RCAG. Conversely, guardians who owned cars, acquired dogs as puppies, lived in households with a maximum of four residents, cared for a sterilized dog or thought that caring for a dog was easier than (or as) expected, had a higher chance of showing adequate adherence to RCAG. The results verify that the socioeconomic and behavioral characteristic of guardians must be taken into consideration for understanding the adherence to RCAG. In addition, it is important to facilitate access to veterinary services and to raise awareness about the significance of a safe and healthy environment for companion animals.