Molecular detection and characterization of SARS-CoV-2 in cats and dogs of positive owners during the first COVID-19 wave in Brazil.

Despite previous reports of SARS-CoV-2 infection in dogs and cats worldwide, the type of swab sample used for its detection through RT-qPCR needs to be better compared and described. Accordingly, as part of a multicenter study in Brazil, the aim of the present study was to assess which rectal or oropharyngeal swabs would be more appropriate for detecting SARS-CoV-2 in cats and dogs, through viral load comparison. Pets of owners diagnosed with COVID-19 in the last 7 days were eligible. A total of 148 animals from four of the five Brazilian geographical regions were analyzed, among which 10/48 cats (20.83%) and 11/100 dogs (11.00%) were positive. The results suggested that oropharyngeal swabs should be considered for SARS-CoV-2 detection, particularly in cats, due to the higher cDNA viral load. Also, the genomic results showed similarities between SARS-CoV-2 animal variants and human variants that were circulating at the time of sampling, thus corroborating the existence of zooanthroponotic transmission. In conclusion, the present study highlighted the importance of SARS-CoV-2 monitoring among cats and dogs, as virus modification may indicate the possibility of mutations in animals and spillover back to owners. Thus, positive individuals should always self-isolate from their pets during COVID-19, to prevent trans-species transmission and mutation.

Hair cortisol concentration, disease severity and quality of life in dogs with atopic dermatitis during lokivetmab therapy.

BACKGROUND: There are no studies which evaluate hair cortisol as a biological marker of stress and anxiety in pruritic dogs during atopic dermatitis therapy. OBJECTIVES: A longitudinal evaluation of hair cortisol concentrations, the severity of disease and the QoL in dogs with cAD during therapy with lokivetmab. ANIMALS: Ten client-owned dogs with cAD. MATERIALS AND METHODS: Dogs were assessed at three time points: at the initial visit at day (D) 0 and at D28, when lokivetmab (2.2-3.2 mg/kg) was administered, and at D56 for one further evaluation. At all time points, pruritus and lesion severity was assessed using the pruritus Visual Analog Scale (PVAS) and Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04). Dog owners filled out a validated QoL questionnaire and hair cortisol concentrations were measured from samples collected from the same area on each dog. RESULTS: There was a significant reduction in PVAS (p < 0.001) and improvement in QoL of dogs (QoL1) and owners (QoL2) after lokivetmab administration, with a positive correlation of the PVAS with QoL1 and QoL2 (r = 0.71 and 0.52, respectively). There was no difference in CADESI-04 scores at the different time points (p = 0.515). A significant reduction in hair cortisol levels at D56 was measured compared with D28 (p < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: Hair cortisol may be a useful marker of stress in dogs with cAD. These results highlight the negative impact of cAD on the QoL of dogs and their owners, and the positive benefit of lokivetmab therapy.

Infection of SARS-CoV-2 in domestic dogs associated with owner viral load.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive pet owners are reported to be a risk factor for infection of their pets; however, the influence of the viral load and associated risks has not been fully established. This study aimed to assess potential association of viral load in owners with the presence of SARS-CoV-2 infection in their dogs. Of 20 SARS-CoV-2-positive pet owners from 13 families in Curitiba, Brazil, 5 of 22 (22.7%) dogs were positive for SARS-CoV-2. Viral presence was detected in oropharyngeal samples for 2 of 5 (40.0%) dogs at 8 and 9 days after the first positive sample. Detection of SARS-CoV-2 in these dogs was associated with higher viral loads in the owners and close owner contact. All 5 RT-qPCR-positive dogs had antibodies to at least one viral protein tested in the serological assay. Molecular detection of SARS-CoV-2 in dogs was statistically associated with clinical signs in owners such as cold, cough, or diarrhea (P = 0.039), number of positive persons in the household (P = 0.002), and higher viral load (P = 0.039). Such findings serve as a warning for risks of human to dog infection, mainly due to sharing beds and other close interactions without protection. In conclusion, people with coronavirus disease 2019 (COVID-19), particularly in households with multiple residents and high viral load, should take the same preventive measures when interacting with their dogs during self-isolation as they do with people.

Report of the presence of Leishmania infantum in the milk of a naturally infected female dog in Brazil.

Dogs are the most important reservoir of Leishmania infantum, the causal agent of visceral leishmaniasis in Brazil. Although lymphoid tissue is the most important biological tissue where amastigotes can be found, this paper describes the presence of L. infantum DNA in the milk of a lactating naturally infected female dog. This finding suggests the need for further studies to elucidate whether breastfeeding can be a route of infection.

Persistent SARS-CoV-2 antigen presence in multiple organs of a naturally infected cat from Brazil.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the disease coronavirus 2019 (COVID-19) in humans. SARS-CoV-2 has been identified in cats with or without clinical signs. Case presentation: We describe the pathological and molecular findings in a six-month-old asymptomatic cat with SARS-CoV-2 infection from Brazil, belonging to a human family with COVID-19 cases. The pool of nasopharynx and oropharynx swabs at day zero tested positive by RT-qPCR for SARS-CoV-2. No amplification resulted from molecular testing performed on days 7 and 14. The cat was hit by a car and died 43 days after the molecular diagnosis. Immunohistochemistry at post-mortem examination demonstrated nucleocapsid protein in samples from the lungs, kidneys, nasal conchae, trachea, intestine, brain and spleen. Conclusion: The present study has highlighted the possibility that viral antigens can be detected by immunohistochemistry in multiple organs six weeks after infection, although the same tissues tested negative by RT-PCR.

Retrospective surveillance of severe acute respiratory syndrome coronavirus 2 in pets from Brazil.

BACKGROUND AND AIM: The emerging concerns regarding the new Coronavirus's ability to cause infection in pets has led to animal testing and worrisome findings reported all over the world in domesticated and wild animals. This study aimed to investigate severe acute respiratory syndrome coronavirus (SARS-CoV)-2 by quantitative reverse transcription-polymerase chain reaction in dog and cat samples with the clinical presentation for respiratory or gastrointestinal disease in Brazil. MATERIALS AND METHODS: One hundred and twenty-five samples were collected from 12 states of Brazil that originated from the gastrointestinal, upper respiratory tract, and other sites, including some pools of samples from before the onset of the pandemic including blood and/or urine samples. They were tested for RT-PCR detection of respiratory or gastrointestinal pathogens through Respiratory or Diarrhea RT-PCR Panels in the TECSA (Tecnologia em Saninade Animal - Animal Health Technology) Veterinary Medicine Laboratory. This work was conducted in compliance with ethical standards. RESULTS: Seven different microorganisms that can cause respiratory and/or gastrointestinal clinical signs were detected in cats (Feline Coronavirus [FCoV], Feline Parvovirus, Feline Leukemia Virus, Feline Calicivirus, Mycoplasma felis, Campylobacter spp., and Cryptosporidium spp.) and three in dogs (canine distemper virus, Cryptosporidium spp., and Babesia spp.). CONCLUSION: Although the samples corresponded to the beginning of coronavirus disease-19 spread in Brazil and clinically correlated with the expected viral replication sites, none of the animals tested positive for SARS-CoV-2; reassuringly, four cats tested positive or FCoV none of them were positive for SARS-CoV2. The epidemiological surveillance of SARS-CoV-2 in pets is considered a one health issue, important for monitoring the disease evolution, spread and minimizing the animal-human health impacts, and directing Public Health Policies.

First report of severe acute respiratory syndrome coronavirus 2 detection in two asymptomatic cats in the state of Pernambuco, Northeastern Brazil.

BACKGROUND AND AIM: Despite worldwide case reports, including Brazilian cases, no frequency study on infection of pets by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been conducted to date in Brazil. Accordingly, the present study was aimed to assess dogs and cats belonging to positive owners in Recife, Northeastern Brazil. MATERIALS AND METHODS: This was a longitudinal prospective study on dogs and cats in the city of Recife whose owners were in isolation at home due to a confirmed laboratory diagnosis of SARS-CoV-2 through reverse-transcriptase polymerase chain reaction (RT-qPCR). Oral and rectal swabs from the pets were tested for the presence of SARS-CoV-2-specific RNA by means of RT-qPCR. RESULTS: Among the pets tested, 0/16 dogs and 2/15 cats were positive for SARS-CoV-2. Interestingly, the two positive cats were owned by two unrelated asymptomatic veterinary students, which, therefore, post a warning to veterinarians worldwide. CONCLUSION: The findings herein indicate that cats may act as sentinels for human cases, particularly sharing households with asymptomatic human cases. Although with small sampling and convenient recruiting, the presence of infected cats by SARS-CoV-2 was most likely due to close cat-human contact with positive owners, posting a human-animal health threat when pets share the same bed and interact with owners without protection, particularly during owner self-isolation. Thus, infected owners should follow the same human preventive guidelines with their pets to avoid spreading infection.

Multi-targeted gene silencing strategies inhibit replication of Canine morbillivirus.

BACKGROUND: Canine morbilivirus (canine distemper virus, CDV) is a highly contagious pathogen associated with high morbidity and mortality in susceptible carnivores. Although there are CDV vaccines available, the disease poses a huge threat to dogs and wildlife hosts due to vaccine failures and lack of effective treatment. Thus, the development of therapeutics is an urgent need to achieve rapid outbreak control and reduce mortality in target species. Gene silencing by RNA interference has emerged as a promising therapeutic approach against different human and animal viruses. In this study, plasmid-based short hairpin RNAs (shRNAs) against three different regions in either CDV nucleoprotein (N), or large polymerase (L) genes and recombinant adenovirus-expressing N-specific multi-shRNAs were generated. Viral cytopathic effect, virus titration, plaque-forming unit reduction, and real-time quantitative RT-PCR analysis were used to check the efficiency of constructs against CDV. RESULTS: In CDV-infected VerodogSLAM cells, shRNA-expressing plasmids targeting the N gene markedly inhibited the CDV replication in a dose-dependent manner, with viral genomes and titers being decreased by over 99%. Transfection of plasmid-based shRNAs against the L gene displayed weaker inhibition of viral RNA level and virus yield as compared to CDV N shRNAs. A combination of shRNAs targeting three sites in the N gene considerably reduced CDV RNA and viral titers, but their effect was not synergistic. Recombinant adenovirus-expressing multiple shRNAs against CDV N gene achieved a highly efficient knockdown of CDV N mRNAs and successful inhibition of CDV replication. CONCLUSIONS: We found that this strategy had strong silencing effects on CDV replication in vitro. Our findings indicate that the delivery of shRNAs using plasmid or adenovirus vectors potently inhibits CDV replication and provides a basis for the development of therapeutic strategies for clinical trials.

Revisiting Key Entry Routes of Human Epidemic Arboviruses into the Mainland Americas through Large-Scale Phylogenomics.

The rapid worldwide spread of chikungunya (CHIKV), dengue (DENV), and Zika (ZIKV) viruses have raised great international concern. Knowledge about the entry routes and geographic expansion of these arboviruses to the mainland Americas remain incomplete and controversial. Epidemics caused by arboviruses continue to cause socioeconomic burden globally, particularly in countries where vector control is difficult due to climatic or infrastructure factors. Understanding how the virus circulates and moves from one country to another is of paramount importance to assist government and health officials in anticipating future epidemics, as well as to take steps to help control or mitigate the spread of the virus. Through the analyses of the sequences of arbovirus genomes collected at different locations over time, we identified patterns of accumulated mutations, being able to trace routes of dispersion of these viruses. Here, we applied robust phylogenomic methods to trace the evolutionary dynamics of these arboviruses with special focus on Brazil, the epicenter of these triple epidemics. Our results show that CHIKV, DENV-1-4, and ZIKV followed a similar path prior to their first introductions into the mainland Americas, underscoring the need for systematic arboviral surveillance at major entry points of human population movement between countries such as airports and seaports.

Latent bovine herpesvirus 1 and 5 in milk from naturally infected dairy cattle.

Bovine herpesvirus 1 and 5 (BoHV-1 and -5) are antigenically and genetically related and can establish latent infection. We aimed to analyze the applicability of the milk sample to detect latently BoHV-infected cattle. BoHV-1 non-vaccinated clinically healthy cows from five dairy cattle herds (herd 1, n=24; herd 2, n=39; herd 3, n=39; herd 4, n=36; herd 5, n=70) were studied. We confirmed the presence of BoHV-1, and for the first time, BoHV-5 in the milk of naturally infected dairy cattle.

The thiopurine nucleoside analogue 6-methylmercaptopurine riboside (6MMPr) effectively blocks Zika virus replication.

Since the emergence of Zika virus (ZIKV) in Brazil in 2015, 48 countries and territories in the Americas have confirmed autochthonous cases of disease caused by the virus. ZIKV-associated neurological manifestations and congenital defects make the development of safe and effective antivirals against ZIKV of utmost importance. Here we evaluated the antiviral activity of 6-methylmercaptopurine riboside (6MMPr), a thiopurine nucleoside analogue derived from the prodrug azathioprine, against the epidemic ZIKV strain circulating in Brazil. In all of the assays, an epithelial (Vero) and a human neuronal (SH-SY5Y) cell line were used to evaluate the cytotoxicity and effective concentrations of 6MMPr against ZIKV. Levels of ZIKV-RNA, viral infectious titre and the percentage of infected cells in the presence or absence of 6MMPr were used to determine antiviral efficacy. 6MMPr decreased ZIKV production by >99% in both cell lines in a dose- and time-dependent manner. Interestingly, 6MMPr was 1.6 times less toxic to SH-SY5Y cells compared with Vero cells, presenting a 50% cytotoxic concentrations (CC50) of 460.3 µM and 291 µM, respectively. The selectivity index of 6MMPr for Vero and SH-SY5Y cells was 11.9 and 22.7, respectively, highlighting the safety profile of the drug to neuronal cells. Taken together, these results identify, for the first time, the thiopurine nucleoside analogue 6MMPr as a promising antiviral candidate against ZIKV that warrants further in vivo evaluation.

6-methylmercaptopurine riboside, a thiopurine nucleoside with antiviral activity against canine distemper virus in vitro.

BACKGROUND: Canine distemper (CD) is a widespread infectious disease that can severely impact a variety of species in the order Carnivora, as well as non-carnivore species such as non-human primates. Despite large-scale vaccination campaigns, several fatal outbreaks have been reported in wild and domestic carnivore populations. This, in association with expansion of the disease host range and the development of vaccine-escape strains, has contributed to an increased demand for therapeutic strategies synergizing with vaccine programs for effectively controlling canine distemper. 6-methylmercaptopurine riboside (6MMPr) is a modified thiopurine nucleoside with known antiviral properties against certain RNA viruses. METHODS: We tested the inhibitory effects of 6MMPr against a wild-type CDV strain infection in cell culture. We measured infectious particle production and viral RNA levels in treated and untreated CDV-infected cells. Ribavirin (RIB) was used as a positive control. RESULTS: Here, we report for the first time the antiviral effects of 6MMPr against canine distemper virus (CDV) in vitro. 6MMPr was able to reduce viral RNA levels and to inhibit the production of infectious CDV particles. The therapeutic selectivity of 6MMPr was approximately six times higher than that of ribavirin. CONCLUSION: Our results indicate that 6MMPr has high anti-CDV potential and warrants further testing against other paramyxoviruses, as well as clinical testing of the compound against CDV.

Campanulotes compar (Burmeister, 1838) (Phthiraptera: Ischnocera) in chickens (Gallus gallus domesticus) from Rio Grande do Norte State, Brazil. The reemergence of an ectoparasite?

The importance of ectoparasites in the transmission of pathogens, as well as the variability of species from one region to another, motivated this notification of the ectoparasite lice Campanulotes compar in range chickens (Gallus gallus domesticus L.) reared in an extensive system in the city of Apodi, Rio Grande do Norte state, in the Northeast region of Brazil. The examined birds were infested with ten males and six females of C. compar. Thus, C. compar is recorded as parasitizing chickens in the state of Rio Grande do Norte after 77 years from its unique citation in the Southeast region of Brazil. We further discuss the possible risks of this finding.

Verification of natural infection of peridomestic rodents by PCV2 on commercial swine farms.

The porcine circovirus-2 (PCV2) is the main agent responsible for porcine circovirus associated diseases (PCVAD). Few studies have been done regarding PCV2 infection in other species. The purpose of this study was to investigate the occurrence of PCV2 infection in the peridomestic rodent species Mus musculus and Rattus rattus on commercial pig farms in Brazil. Immunohistochemistry assay demonstrated PCV2 in the spleen, lung and kidney. Viral DNA was detected in tissues by nested PCR assay. Partial sequences of PCV2 genomes detected in the rodents had strong identity with gene sequences of PCV2 isolates from pigs. These results show that the studied peridomestic rodent species can be naturally infected by PCV2. However, further studies are needed to confirm PCV2 transmission from rodents to pigs.