Herpesvirus, Flavivirus, and Coronavirus Surveillance in Magnificent Frigatebirds (Fregata magnificens), Alcatrazes Archipelago, Southeastern Brazil.

We surveyed the presence of herpesvirus, flavivirus, and coronavirus in 20 Magnificent Frigatebirds (Fregata magnificens) from the protected Alcatrazes Island, Alcatrazes archipelago, Brazil. One adult female was positive for herpesvirus (5% occurrence; 95% confidence interval -5.5 to 15.5), whereas none of the samples were PCR-positive for flavivirus or coronavirus. The obtained herpesvirus was highly similar to the one responsible for annual mortality of Magnificent Frigatebird chicks on Grand Connétable Island, French Guiana; however, no episodes of mass mortality have been recorded in the birds from Alcatrazes. Our findings indicate that this virus may be widespread in Magnificent Frigatebirds of the southwestern Atlantic. The observed differences in morbidity and mortality may be the result of basal immunosuppression of the birds from French Guiana related to environmental or nutritional conditions. The Alcatrazes archipelago sustains the largest frigatebird breeding colony of the southern Atlantic; future monitoring studies with larger sampling sizes are needed to further determine the epidemiologic relevance of the detected herpesviruses, as well as other viruses (e.g., flaviviruses, coronaviruses, avian influenza virus), in seabirds of Alcatrazes Island.

Geographic Distribution of Common Vampire Bat Desmodus rotundus (Chiroptera: Phyllostomidae) Shelters: Implications for the Spread of Rabies Virus to Cattle in Southeastern Brazil.

Desmodus rotundus bats show a complex social structure and developed adaptive characteristics, considered key features of a pathogen disseminator, such as the rabies virus, among bats and other mammals, including cattle and humans. Our aim was to understand the correlation between the environment and the ecological features of these bats in bovine rabies outbreaks. Geostatistical analyses were performed, covering 104 cattle positives for rabies, between 2016 and 2018, in 25 municipalities, in addition to the characteristics of D. rotundus colonies mapped during this period in the state of São Paulo, Brazil. Data from the shelters showed that 86.15% were artificial, mainly abandoned houses (36.10%) and manholes (23.87%), in addition to demonstrating a correlation between these shelters and a higher concentration of bovine rabies cases. Due to their adaptive capacity, these bats choose shelters close to the food source, such as livestock. In Brazil, D. rotundus is the main transmitter of rabies and the cause of outbreaks in cattle and deaths in humans, considering the advance of humans in previously preserved ecosystems. There seems to be a correlation between the impact of anthropic changes on the environment, mainly for the expansion of pasture for cattle and the outbreaks of bovine rabies in this area.

The order of events on Avian coronavirus life cycle shapes the order of quasispecies evolution during host switches.

To understand the population genetics events during coronavirus host switches, the Beaudette strain of Avian coronavirus (AvCoV) adapted to BHK-21 cells was passaged 15 times in VERO cells, the virus load and the variants at each passage being determined by RT-qPCR and genome-length deep sequencing. From BHK-21 P2 to VERO P3, a trend for the extinction of variants was followed by stability up to VERO P11 and both the emergence and the rise in frequency in some variants, while the virus loads were stable up to VERO P12. At the spillover from BHK-21 to VERO cells, variants that both emerged, showed a rise in frequency or were extinguished were detected on the spike, while variants at the M gene showed the same pattern only at VERO passage 13. Furthermore, nsps 3-5, 9 and 15 variants were detected at lower passages compared to the consensus sequences, with those at nsp3 being detected in the spectra also at higher passages. This suggests that quasispecies coronavirus evolution in spillovers follows the virus life cycle, starting with the evolution of the receptor binding proteins, followed by the replicase and then proteins involved in virion assembly, keeping the general fitness of the mutant spectrum stable.

Genomic Analysis of Novel Poxvirus Brazilian Porcupinepox Virus, Brazil, 2019.

We obtained the complete sequence of a novel poxvirus, tentatively named Brazilian porcupinepox virus, from a wild porcupine (Coendou prehensilis) in Brazil that had skin and internal lesions characteristic of poxvirus infection. The impact of this lethal poxvirus on the survival of this species and its potential zoonotic importance remain to be investigated.

Emergence of Avian coronavirus genotype GI-11 in Colombia.

Avian coronavirus (AvCoV/IBV) is a virus with high morbidity, which can cause respiratory, digestive, renal, and reproductive diseases in chickens. Molecular detection and sequencing are the main tool for identification and classification of AvCoV. Thirty-six samples were collected in three broiler farms from different regions in Colombia, due to mortality increase; ten samples were positive using RT-qPCR targeted to the 5' UTR of AvCoV, and one sample was positive and had its partial S gene sequenced. Phylogenetic analysis revealed that this strain belongs to the GI-11 lineage, similar to the Brazilian cluster. Several lineages have already been described in Colombia but, to the best of our knowledge, this is the first time that GI-11 has been detected in this country, which suggests that this subtype may be more widespread in South America than previously thought.

Near-Complete Genome Sequence of Feline Immunodeficiency Virus from Colombia.

Feline immunodeficiency virus (FIV) is an important pathogen of domestic and wild felids. Although serological tests suggest the presence of FIV in cats from Colombia, no molecular characterization has been reported. Here, we describe the near-complete genome of FIV subtype A from a Colombian domestic cat.

Rabies virus diversification in aerial and terrestrial mammals.

Rabies is a fatal zoonotic infection of the central nervous system of mammals and has been known to humans for millennia. The etiological agent, is a neurotropic RNA virus in the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus. There are currently accepted to be two cycles for rabies transmission: the urban cycle and the sylvatic cycle. The fact that both cycles originated from a common RABV or lyssavirus ancestor and the adaptive divergence that occurred since then as this ancestor virus adapted to a wide range of fitness landscapes represented by reservoir species in the orders Carnivora and Chiroptera led to the emergence of the diverse RABV lineages currently found in the sylvatic and urban cycles. Here we study full genome phylogenies and the time to the most recent common ancestor (TMRCA) of the RABVs in the sylvatic and urban cycles. Results show that there were differences between the nucleotide substitution rates per site per year for the same RABV genes maintained independently in the urban and sylvatic cycles. The results identify the most suitable gene for phylogenetic analysis, heterotachy among RABV genes and the TMRCA for the two cycles.

Intracerebral transfection of anti-rabies virus antibodies is an effective therapy for rabies.

Rabies is a neurological disease with 100% lethality. Some of the rare human patients who survived after multiple drug treatment had severe sequelae. The present study showed that after 48 h of RABV inoculation, mice injected intracerebrally with anti-RABV F (ab')2 plus Bioporter® showed 70% survival compared to the control group, suggesting that transfection of anti-RABV antibodies to the brain may prevent or delay the spread of RABV at an early stage of infection. This result may provide important protocol results in intracellular antibody delivery to prevent the fatal outcome of the disease.

Genome constellations of rotavirus a isolated from avian species in Brazil, 2008-2015.

Rotaviruses are members of the family Reoviridae and are a common cause of acute diarrhea in many mammalian and avian species. They are non-enveloped icosahedral particles and their genome comprises 11 segments of double-stranded RNA, which encodes six structural proteins (VP1-4, VP6-7) and six nonstructural proteins (NSP1-6). Genotypes are defined based upon the diversity found in these genes and viral characterization plays a central role on epidemiological studies and prevention. Here we investigate the distribution of Brazilian RVAs genotypes in 8 chicken samples collected between 2008 and 2015 from different regions by RT-PCR, partial (Sanger) nucleotide sequencing and phylogenetic analysis from all rotavirus genes. Although the identified genotypes were typical from avian host species, when analyzed together, they form novel genetic constellations: G19-P[31]-I11-R6-C6-M7-A16-N6-T8-E10-H8 and G19-P[31]-I4-R4-C4-M4-A16-N4-T4-E4-H4. This study highlights that avian rotaviruses are widespread among commercial farms in Brazil, and the co-circulation of at least two different genomic constellations indicates that may present a way bigger genetic variability, that can be increased by the possible transmission events from other birds, lack of specific preventive measures, as well as the different viral evolution mechanisms.

Extinction and emergence of genomic haplotypes during the evolution of Avian coronavirus in chicken embryos.

Avian coronavirus (AvCoV) is ubiquitously present on poultry as a multitude of virus lineages. Studies on AvCoV phenotypic traits are dependent on the isolation of field strains in chicken embryonated eggs, but the mutant spectrum on each isolate is not considered. This manuscript reports the previously unknown HTS (high throughput sequencing)-based complete genome haplotyping of AvCoV isolates after passages of two field strains in chicken embryonated eggs. For the first and third passages of strain 23/2013, virus loads were 6.699 log copies/ µL and 6 log copies/ µL and, for 38/2013, 5.699 log copies/µL and 2.699 log copies/µL of reaction, respectively. The first passage of strain 23/2013 contained no variant haplotype, while, for the third passage, five putative variant haplotypes were found, with > 99.9% full genome identity with each other and with the dominant genome. Regarding strain 38/2013, five variant haplotypes were found for the first passage, with > 99.9% full genome identity with each other and with the dominant genome, and a single variant haplotype was found. Extinction and emergence of haplotypes with polymorphisms in genes involved in receptor binding and regulation of RNA synthesis were observed, suggesting that phenotypic traits of AvCoV isolates are a result of their mutant spectrum.

Influence of early use of antimicrobial on the health and performance of Holstein calves in the first month of life / Influência do uso precoce de antimicrobiano na sanidade de bezerras holandesas no primeiro mês de vida

The early use of antimicrobial therapy has been introduced in many farms to prevent diarrhea and respiratory disease in young calves; however, there is controversy about whether this practice has a beneficial effect on the health of these animals. This study evaluated the influence of the early use of antimicrobials on the health and performance of neonatal Holstein calves. Twenty-six Holstein calves were screened and divided into two groups, according to the administration (ATB+), or not (ATB-) of tulathromycin (2.5mg/kg, subcutaneously) within the first 12 hours of life. Calves were evaluated by general clinical examination, fecal score, respiratory score, and external palpation of the umbilical region, besides fecal output of dry matter. Anemia was determined by using an automatic system and, also, using a commercial kit for iron dosage. Diarrhea was diagnosed by a centrifuge-flotation technique using a sugar solution (Cryptosporidium) and multiplex semi-nested RT-PCR (rotavirus/coronavirus). The performance of the calves was estimated by Daily Weight Gain (DWG). The young dairy calves were evaluated within 12 hours of birth (≤12h) and at 3-5th (D3-5), 7-9th (D7-9), 13-15th (D13-15), 20-23rd (D20-23), and 27-30th (D27-30) days of life. No difference was noted between the ATB+ and ATB- groups concerning heart rate, respiratory frequency, and rectal temperature. Erythrogram showed a higher frequency of anemia in ATB- group (P=0.016) at the D3-5 check-up; lower values of serum iron were also observed simultaneously (P=0.051). Thirteen cases of respiratory disease were detected during this study; however, no significant difference was observed between the groups in this regard. The frequency of diarrhea (fecal score 2-3) was high in both groups, peaking at D13-D15. No differences were noted between the groups regarding the frequency of diarrhea when considering the dry fecal matter. The predominant etiological agent for diarrhea was Cryptosporidium spp.. The DWG was similar between groups, with maximum weight reduction on D13-15. The administration of tulathromycin in prophylactic dose (2.5mg/kg) at birth decreased the frequency of anemia but did not influence weight gain or the prevalence of diarrhea.(AU)

O uso precoce de antimicrobianos tem sido adotado em muitas fazendas para profilaxia das diarreias e doença respiratória em bezerras, no entanto existem controvérsias sobre os beneficios desta prática na saúde desses animais. Esta pesquisa avaliou a influência do uso precoce de antimicrobiano na sanidade e desempenho de bezerras holandesas recém-nascidas. Para tanto foram selecionadas 26 bezerras Holandesas distribuídas de acordo com a aplicação (ATB+) ou não (ATB-) de tulatromicina (2,5mg/Kg) por via subcutânea até 12h de vida. As bezerras foram examinadas por meio de exame clínico geral, escore fecal, escore respiratório e palpação externa da região umbilical, além da matéria seca fecal. A presença de anemias foi determinada pelo eritrograma utilizando sistema automático e além da dosagem de ferro utilizando kit comercial. O diagnóstico etiológico das diarreias foi investigado por meio da técnica de flutuação em solução saturada de sacarose (Cryptosporidium) e multiplex semi-nested RT-PCR (rotavírus/coronavírus). O desempenho das bezerras foi estimado pelo ganho de peso. As bezerras foram avaliadas até doze horas após o nascimento (≤12h); 3-5º (D3-5); 7-9º (D7-9); 13-15º (D13-15); 20-23º (D20-23); e 27-30º dias de vida (D27-30). Não foram encontradas diferenças entre os grupos ATB+ e ATB- em relação à frequência cardíaca, frequência respiratória e temperatura retal. O eritrograma revelou maior frequência de anemias no grupo ATB- (P=0,016) no D3-5. Neste momento também foram observados menores valores de ferro sérico (P=0,051). Foram detectados treze casos de doença respiratória durante o estudo, no entanto não foi possível detectar diferença entre os grupos. A frequência de diarreias (escore fecal 2 e 3) foi alta em ambos os grupos, observando-se pico no D13-15 (ATB+=92,3%; ATB-=92,3%). Não observamos diferenças entre os grupos em relação a frequência de diarreia considerando-se a matéria seca fecal. O agente etiológico predominante nas diarreias foi o Cryptosporidium. O ganho de peso diário foi igual entre grupos, com intensa redução no GPD no D13-15. A administração de tulatromicina na dose profilática (2,5mg/Kg) ao nascimento diminuiu a frequência de anemias e não influenciou no ganho de peso e prevalência de diarreias.(AU)

Complete Genome Sequence of Avian Coronavirus Strain D274.

Avian coronavirus, the causative agent of avian infectious bronchitis, occurs as multiple genotypes and lineages, and full genomes are not available for the majority of them. This paper reports the (previously unknown) complete genome sequence of strain D274 of this virus (27,599 nucleotides), isolated from chickens in The Netherlands in 1979.

Genetic diversity of BCoV in Brazilian cattle herds.

Bovine coronavirus (BCoV) is one of the main aetiological agents of gastroenteritis in calves, causing significant economic damage to livestock. This study aims to characterise BCoV genetically on the basis of the N gene. A total of 114 faecal samples from beef and dairy calves with or without clinical symptoms of diarrhoea from five Brazilian states (São Paulo, Minas Gerais, Santa Catarina, Mato Grosso and Bahia) were evaluated between 2008 and 2015 by technique of Semi-nested RT-PCR for gene N and genealogical analysis. Of the 114 samples analysed, 14.91% (17/114) were positive. BCoV was detected in 22.72% (10/44) of the animals with diarrhoea and in 10% (7/70) of asymptomatic animals. BCoV was identified in calves from rural properties located in all of the regions sampled. Genealogical analysis showed that the Brazilian sequences of BCoV for the gene which codes for the N protein can be broken down into two distinct clusters, and the samples from this study were closely linked to Asian strains. These results contribute to the molecular characterization of BCoV in Brazil and are the first report of the circulation of BCoV in the states of Santa Catarina and Bahia.

Complete Genome Sequencing of Mycobacterium bovis SP38 and Comparative Genomics of Mycobacterium bovis and M. tuberculosis Strains.

Mycobacterium bovis causes bovine tuberculosis and is the main organism responsible for zoonotic tuberculosis in humans. We performed the sequencing, assembly and annotation of a Brazilian strain of M. bovis named SP38, and performed comparative genomics of M. bovis genomes deposited in GenBank. M. bovis SP38 has a traditional tuberculous mycobacterium genome of 4,347,648 bp, with 65.5% GC, and 4,216 genes. The majority of CDSs (2,805, 69.3%) have predictive function, while 1,206 (30.07%) are hypothetical. For comparative analysis, 31 M. bovis, 32 M. bovis BCG, and 23 Mycobacterium tuberculosis genomes available in GenBank were selected. M. bovis RDs (regions of difference) and Clonal Complexes (CC) were identified in silico. Genome dynamics of bacterial groups were analyzed by gene orthology and polymorphic sites identification. M. bovis polymorphic sites were used to construct a phylogenetic tree. Our RD analyses resulted in the exclusion of three genomes, mistakenly annotated as virulent M. bovis. M. bovis SP38 along with strain 35 represent the first report of CC European 2 in Brazil, whereas two other M. bovis strains failed to be classified within current CC. Results of M. bovis orthologous genes analysis suggest a process of genome remodeling through genomic decay and gene duplication. Quantification, pairwise comparisons and distribution analyses of polymorphic sites demonstrate greater genetic variability of M. tuberculosis when compared to M. bovis and M. bovis BCG (p ≤ 0.05), indicating that currently defined M. tuberculosis lineages are more genetically diverse than M. bovis CC and animal-adapted MTC (M. tuberculosis Complex) species. As expected, polymorphic sites annotation shows that M. bovis BCG are subjected to different evolutionary pressures when compared to virulent mycobacteria. Lastly, M. bovis phylogeny indicates that polymorphic sites may be used as markers of M. bovis lineages in association with CC. Our findings highlight the need to better understand host-pathogen co-evolution in genetically homogeneous and/or diverse host populations, considering the fact that M. bovis has a broader host range when compared to M. tuberculosis. Also, the identification of M. bovis genomes not classified within CC indicates that the diversity of M. bovis lineages may be larger than previously thought or that current classification should be reviewed.

First Nearly Complete Genome Sequence of Feline immunodeficiency virus from Brazil.

Feline immunodeficiency virus (FIV) has worldwide distribution; nevertheless, only a few FIV genomes from domestic cats are available. This is the first report of a nearly complete genome of FIV from a Brazilian cat (8,967 nucleotides [nt]), including the entire coding region and the 3' untranslated region.

Molecular detection of viral agents in free-ranging and captive neotropical felids in Brazil.

We describe molecular testing for felid alphaherpesvirus 1 (FHV-1), carnivore protoparvovirus 1 (CPPV-1), feline calicivirus (FCV), alphacoronavirus 1 (feline coronavirus [FCoV]), feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and canine distemper virus (CDV) in whole blood samples of 109 free-ranging and 68 captive neotropical felids from Brazil. Samples from 2 jaguars ( Panthera onca) and 1 oncilla ( Leopardus tigrinus) were positive for FHV-1; 2 jaguars, 1 puma ( Puma concolor), and 1 jaguarundi ( Herpairulus yagouaroundi) tested positive for CPPV-1; and 1 puma was positive for FIV. Based on comparison of 103 nucleotides of the UL24-UL25 gene, the FHV-1 sequences were 99-100% similar to the FHV-1 strain of domestic cats. Nucleotide sequences of CPPV-1 were closely related to sequences detected in other wild carnivores, comparing 294 nucleotides of the VP1 gene. The FIV nucleotide sequence detected in the free-ranging puma, based on comparison of 444 nucleotides of the pol gene, grouped with other lentiviruses described in pumas, and had 82.4% identity with a free-ranging puma from Yellowstone Park and 79.5% with a captive puma from Brazil. Our data document the circulation of FHV-1, CPPV-1, and FIV in neotropical felids in Brazil.

Complete Genome of Avian coronavirus Vaccine Strains Ma5 and BR-I.

Avian coronavirus (AvCoV) is a ubiquitous multiple-serotype pathogen of poultry, and its control is mainly based on the use of vaccines. We report here the previously unknown full genomes of the Ma5 (27,652 nucleotides [nt]) and BR-I (27,618 nt) AvCoV vaccine strains of the GI-1 (Massachusetts) and GI-11 (Brazil) types.

Report of the first clinical case of intestinal trichomoniasis caused by Tritrichomonas foetus in a cat with chronic diarrhoea in Brazil.

BACKGROUND: Tritrichomonas foetus is an emergent and important enteric pathogen of cats, which causes prolonged diarrhoea in cats. CASE PRESENTATION: This study describes a T. foetus infection in a seven-month-old, entire male domestic shorthair kitten with a six-month history of persistent large intestinal diarrhoea, faecal incontinence, prostration, apathy and weight loss. Parasites were microscopically observed and confirmed by PCR and DNA sequencing. Molecular analyses were carried out comparing the sequence obtained in this study with T. foetus and T. suis. Retrieved from GenBank. After treatment with ronidazole, the cat showed resolution of clinical signs. CONCLUSIONS: This is the first clinical case of T. foetus infection in a chronic diarrheic cat in Brazil and South America, confirming the presence of this pathogen in this part of the world and highlighting the importance of this protozoa being considered in the differential diagnosis of cats presenting diarrhoea of the large intestine. Our case report enriches our knowledge on the geographical distribution of T. foetus in cats in Brazil and provides further understanding of the clinical significance of feline intestinal trichomoniasis in this country.

An Atypical Enteropathogenic Escherichia coli Isolate with Possible Human and Domestic Animal Origin from a Free-Living Lear's Macaw (Anodorhynchus leari).

An atypical enteropathogenic Escherichia coli was isolated from an asymptomatic nestling of the endangered Lear's Macaw (Anodorhynchus leari). Phylogenetically, it was identical to bovine and human strains and highly similar to other human and domestic animal isolates. We discuss potential routes of transmission and risks to conservation of this species.