Genome analysis of Streptococcus spp. isolates from animals in pre-antibiotic era with respect to antibiotic susceptibility and virulence gene profiles.

Lyophilized Streptococcus spp. isolates (n = 50) from animal samples submitted to the diagnostic laboratory at the University of Connecticut in the 1940s were revivified to investigate the genetic characteristics using whole-genome sequencing (WGS). The Streptococcus spp. isolates were identified as follows; S. agalactiae (n = 14), S. dysgalactiae subsp. dysgalactiae (n = 10), S. dysgalactiae subsp. equisimils (n = 5), S. uberis (n = 8), S. pyogenes (n = 7), S. equi subsp. zooepidemicus (n = 4), S. oralis (n = 1), and S. pseudoporcinus (n = 1). We identified sequence types (ST) of S. agalactiae, S. dysgalactiae, S. uberis, S. pyogenes, and S. equi subsp. zooepidemicus and reported ten novel sequence types of those species. WGS analysis revealed that none of Streptococcus spp. carried antibiotic resistance genes. However, tetracycline resistance was observed in four out of 15 S. dysgalactiae isolates and in one out of four S. equi subsp. zooepidemicus isolate. This data highlights that antimicrobial resistance is pre-existed in nature before the use of antibiotics. The draft genome sequences of isolates from this study and 426 complete genome sequences of Streptococcus spp. downloaded from BV-BRC and NCBI GenBank database were analyzed for virulence gene profiles and phylogenetic relationships. Different Streptococcus species demonstrated distinct virulence gene profiles, with no time-related variations observed. Phylogenetic analysis revealed high genetic diversity of Streptococcus spp. isolates from the 1940s, and no clear spatio-temporal clustering patterns were observed among Streptococcus spp. analyzed in this study. This study provides an invaluable resource for studying the evolutionary aspects of antibiotic resistance acquisition and virulence in Streptococcus spp.

Simultaneous Deletion of the 9GL and UK Genes from the African Swine Fever Virus Georgia 2007 Isolate Offers Increased Safety and Protection against Homologous Challenge.

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Successful experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFV. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against challenge with parental viruses. Deletion of the 9GL (B119L) gene in the highly virulent ASFV isolates Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Δ9GL viruses) resulted in complete protection when challenged with parental isolates. When similar deletions were created within the ASFV Georgia 2007 (ASFV-G) genome, attenuation was achieved but the protective and lethal doses were too similar. To enhance attenuation of ASFV-G, we deleted another gene, UK (DP96R), which was previously shown to be involved in attenuation of the ASFV E70 isolate. Here, we report the construction of a double-gene-deletion recombinant virus, ASFV-G-Δ9GL/ΔUK. When administered intramuscularly (i.m.) to swine, there was no induction of disease, even at high doses (106 HAD50). Importantly, animals infected with 104 50% hemadsorbing doses (HAD50) of ASFV-G-Δ9GL/ΔUK were protected as early as 14 days postinoculation when challenged with ASFV-G. The presence of protection correlates with the appearance of serum anti-ASFV antibodies, but not with virus-specific circulating ASFV-specific gamma interferon (IFN-γ)-producing cells. ASFV-G-Δ9GL/ΔUK is the first rationally designed experimental ASFV vaccine that protects against the highly virulent ASFV Georgia 2007 isolate as early as 2 weeks postvaccination. IMPORTANCE: Currently, there is no commercially available vaccine against African swine fever. Outbreaks of the disease are devastating to the swine industry and are caused by circulating strains of African swine fever virus. Here, we report a putative vaccine derived from a currently circulating strain but containing two deletions in two separate areas of the virus, allowing increased safety. Using this genetically modified virus, we were able to vaccinate swine and protect them from developing ASF. We were able to achieve protection from disease as early as 2 weeks after vaccination, even when the pigs were exposed to a higher than normal concentration of ASFV.

African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus.

UNLABELLED: African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-ΔMGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-ΔMGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-ΔMGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 10(2) or 10(4) 50% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-ΔMGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. IMPORTANCE: The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer protection in pigs challenged with homologous viruses. Here we have produced an attenuated recombinant ASFV derived from highly virulent ASFV strain Georgia (ASFV-G) lacking only six of the multigene family 360 (MGF360) and MGF505 genes (ASFV-G-ΔMGF). It is demonstrated, by first time, that deleting specific MGF genes alone can completely attenuate a highly virulent field ASFV isolate. Recombinant virus ASFV-G-ΔMGF effectively confers protection in pigs against challenge with ASFV-G when delivered once via the intramuscular (i.m.) route. The protection against ASFV-G is highly effective by 28 days postvaccination. This is the first report of an experimental vaccine that induces solid protection against virulent ASFV-G.

The progressive adaptation of a georgian isolate of African swine fever virus to vero cells leads to a gradual attenuation of virulence in swine corresponding to major modifications of the viral genome.

UNLABELLED: African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified, or cell culture-adapted ASFV have been evaluated, but no commercial vaccine is available to control African swine fever (ASF). We report here the genotypic and phenotypic analysis of viruses obtained at different passages during the process of adaptation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) to grow in cultured cell lines. ASFV-G was successively passaged 110 times in Vero cells. Viruses obtained at passages 30, 60, 80, and 110 were evaluated in vitro for the ability to replicate in Vero cells and primary swine macrophages cultures and in vivo for assessing virulence in swine. Replication of ASFV-G in Vero cells increased with successive passages, corresponding to a decreased replication in primary swine macrophages cultures. In vivo, progressive loss of virus virulence was observed with increased passages in Vero cells, and complete attenuation of ASFV-G was observed at passage 110. Infection of swine with the fully attenuated virus did not confer protection against challenge with virulent parental ASFV-G. Full-length sequence analysis of each of these viruses revealed significant deletions that gradually accumulated in specific areas at the right and left variable ends of the genome. Mutations that result in amino acid substitutions and frameshift mutations were also observed, though in a rather limited number of genes. The potential importance of these genetic changes in virus adaptation/attenuation is discussed. IMPORTANCE: The main problem in controlling ASF is the lack of vaccines. Attempts to produce vaccines by adaptation of ASFV to cultured cell lines have been made. These attempts led to the production of attenuated viruses that conferred only homologous protection. Specifics regarding adaptation of these isolates to cell cultures have been insufficiently described. Details like the numbers of passages required to obtain attenuated viruses, genetic modifications introduced into the virus genomes along passages, and the extent of attenuation and induced protective efficacy are not readily available. In this study, we assessed the changes that lead to decreased growth in swine macrophages and to attenuation in swine. Loss of virulence, probably associated with limited replication in vivo, may lead to the lack of protective immunity in swine observed after challenge. This report provides valuable information that can be used to further the understanding of ASFV gene function, virus attenuation, and protection against infection.

African Swine Fever Virus Georgia 2007 with a Deletion of Virulence-Associated Gene 9GL (B119L), when Administered at Low Doses, Leads to Virus Attenuation in Swine and Induces an Effective Protection against Homologous Challenge.

UNLABELLED: African swine fever virus (ASFV) is the etiological agent of an often lethal disease of domestic pigs. Disease control strategies have been hampered by the unavailability of vaccines against ASFV. Since its introduction in the Republic of Georgia, a highly virulent virus, ASFV Georgia 2007 (ASFV-G), has caused an epizootic that spread rapidly into Eastern European countries. Currently no vaccines are available or under development to control ASFV-G. In the past, genetically modified ASFVs harboring deletions of virulence-associated genes have proven attenuated in swine, inducing protective immunity against challenge with homologous parental viruses. Deletion of the gene 9GL (open reading frame [ORF] B119L) in highly virulent ASFV Malawi-Lil-20/1 produced an attenuated phenotype even when administered to pigs at 10(6) 50% hemadsorption doses (HAD50). Here we report the construction of a genetically modified ASFV-G strain (ASFV-G-Δ9GLv) harboring a deletion of the 9GL (B119L) gene. Like Malawi-Lil-20/1-Δ9GL, ASFV-G-Δ9GL showed limited replication in primary swine macrophages. However, intramuscular inoculation of swine with 10(4) HAD50 of ASFV-G-Δ9GL produced a virulent phenotype that, unlike Malawi-Lil-20/1-Δ9GL, induced a lethal disease in swine like parental ASFV-G. Interestingly, lower doses (10(2) to 10(3) HAD50) of ASFV-G-Δ9GL did not induce a virulent phenotype in swine and when challenged protected pigs against disease. A dose of 10(2) HAD50 of ASFV-G-Δ9GLv conferred partial protection when pigs were challenged at either 21 or 28 days postinfection (dpi). An ASFV-G-Δ9GL HAD50 of 10(3) conferred partial and complete protection at 21 and 28 dpi, respectively. The information provided here adds to our recent report on the first attempts toward experimental vaccines against ASFV-G. IMPORTANCE: The main problem for controlling ASF is the lack of vaccines. Studies on ASFV virulence lead to the production of genetically modified attenuated viruses that induce protection in pigs but only against homologous virus challenges. Here we produced a recombinant ASFV lacking virulence-associated gene 9GL in an attempt to produce a vaccine against virulent ASFV-G, a highly virulent virus isolate detected in the Caucasus region in 2007 and now spreading though the Caucasus region and Eastern Europe. Deletion of 9GL, unlike with other ASFV isolates, did not attenuate completely ASFV-G. However, when delivered once at low dosages, recombinant ASFV-G-Δ9GL induces protection in swine against parental ASFV-G. The protection against ASFV-G is highly effective after 28 days postvaccination, whereas at 21 days postvaccination, animals survived the lethal challenge but showed signs of ASF. Here we report the design and development of an experimental vaccine that induces protection against virulent ASFV-G.

Suburban Population of Bobcats (Lynx rufus) in Connecticut, USA, Tested Negative for SARS-CoV-2, November 2021-February 2022.

A SARS-CoV-2 genomic and serologic survey was performed in a population of bobcats (Lynx rufus) inhabiting the state of Connecticut, USA. Wild animal populations are becoming established in densely populated cities with increased likelihood of direct or indirect contact with humans, as well as with household cats and dogs. Wild-caught bobcats (n=38) tested negative for SARS-CoV-2 genomic RNA by reverse-transcription quantitative PCR and for virus-neutralizing antibodies by ELISA, suggesting that either the species is not susceptible to SARS-CoV-2 or that the surveyed population has not yet been exposed to a source of infectious virus. However, this limited survey cannot rule out that human-to-bobcat or unknown reservoir-to-bobcat transmission of the virus occurs in nature.

The complete coding sequence of a rabies lyssavirus (RABV) detected in an American black bear (Ursus americanus) in Connecticut, USA.

The complete coding sequence of a rabies lyssavirus (RABV) detected in a black bear (Ursus americanus) was generated. RNA extracted from brain tissues was amplified using reverse transcription followed by tiling PCR sequencing to obtain RABV whole viral genome. Sequencing was performed using an Illumina ISeq 100 instrument.

Classical swine fever virus p7 protein is a viroporin involved in virulence in swine.

The nonstructural protein p7 of classical swine fever virus (CSFV) is a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytosolic loop, respectively. Using reverse genetics, partial in-frame deletions of p7 were deleterious for virus growth, demonstrating that CSFV p7 function is critical for virus production in cell cultures. A panel of recombinant mutant CSFVs was created using alanine scanning mutagenesis of the p7 gene harboring sequential three- to six-amino-acid residue substitutions spanning the entire protein. These recombinant viruses allowed the identification of the regions within p7 that are critical for virus production in vitro. In vivo, some of these viruses were partially or completely attenuated in swine relative to the highly virulent parental CSFV Brescia strain, indicating a significant role of p7 in CSFV virulence. Structure-function analyses in model membranes emulating the endoplasmic reticulum lipid composition confirmed that CSFV p7 is a pore-forming protein, and that pore-forming activity resides in the C-terminal transmembrane helix. Therefore, p7 is a viroporin which is clearly involved in the process of CSFV virulence in swine.

Mycobacteriosis, Mycobacterium chelonae, in a captive yellow stingray (Urobatis jamaicensis).

An adult yellow stingray (Urobatis jamaicensis) from a touch-tank exhibit developed a large abscess on the dorsal aspect of the calvarium and swollen soft tissue surrounding the left spiracle. A large amount of fluid exudate was drained from the abscess. Mycobacterium chelonae was diagnosed by cytology of the exudate and by polymerase chain reaction and sequencing. The animal was euthanized and disseminated mycobacteriosis was confirmed with histology.

Whole genome sequencing and phylogenetic analysis of West Nile viruses from animals in New England, United States, 2021.

West Nile virus is a mosquito-borne Flavivirus which is the leading cause of global arboviral encephalitis. We sequenced WNVs from an American crow found in Connecticut and an alpaca found in Massachusetts which were submitted to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL). We report here the complete protein-coding sequences (CDS) of the WNVs (WNV 21-3957/USA CT/Crow/2021 and WNV 21-3782/USA MA/Alpaca/2021) and their phylogenetic relationship with other WNVs recovered from across the United States. In the phylogenetic analysis, the WNVs from this study belonged to the WNV lineage 1. The WNV 21-3957/USA CT/Crow/2021 clustered with WNVs from a mosquito and birds in New York during 2007-2013. Interestingly, the virus detected in the alpaca, WNV 21-3782/USA MA/Alpaca/2021 clustered with WNVs from mosquitos in New York, Texas, and Arizona during 2012-2016. The genetic differences between the viruses detected during the same season in an American crow and an alpaca suggest that vector-host feeding preferences are most likely driving viral transmission. The CDS of the WNVs and their phylogenetic relationships with other WNVs established in this study would be useful as reference data for future investigations on WNVs. Seasonal surveillance of WNV in birds and mammals and the genetic characterization of detected viruses are necessary to monitor patterns of disease presentations and viral evolution within a geographical area.

Evaluation of the Deletion of the African Swine Fever Virus Gene O174L from the Genome of the Georgia Isolate.

African swine fever virus (ASFV) is a structurally complex, double-stranded DNA virus, which causes African swine fever (ASF), a contagious disease affecting swine. ASF is currently affecting pork production in a large geographical region, including Eurasia and the Caribbean. ASFV has a large genome, which harbors more than 160 genes, but most of these genes' functions have not been experimentally characterized. One of these genes is the O174L gene which has been experimentally shown to function as a small DNA polymerase. Here, we demonstrate that the deletion of the O174L gene from the genome of the virulent strain ASFV Georgia2010 (ASFV-G) does not significantly affect virus replication in vitro or in vivo. A recombinant virus, having deleted the O174L gene, ASFV-G-∆O174L, was developed to study the effect of the O174L protein in replication in swine macrophages cultures in vitro and disease production when inoculated in pigs. The results demonstrated that ASFV-G-∆O174L has similar replication kinetics to parental ASFV-G in swine macrophage cultures. In addition, animals intramuscularly inoculated with 102 HAD50 of ASFV-G-∆O174L presented a clinical form of the disease that is indistinguishable from that induced by the parental virulent strain ASFV-G. All animals developed a lethal disease, being euthanized around day 7 post-infection. Therefore, although O174L is a well-characterized DNA polymerase, its function is apparently not critical for the process of virus replication, both in vitro and in vivo, or for disease production in domestic pigs.

Whole genome sequencing and phylogenetic analysis of African swine fever virus detected in a backyard pig in Mongolia, 2019.

African swine fever (ASF) is a highly contagious and fatal disease affecting domestic and wild pigs caused by the African swine fever virus (ASFV). Since the first outbreak in China in August 2018, ASF has spread rapidly in Asia. and the first case in Mongolia was confirmed in January 2019. In this study, we report the first whole genome sequence of an ASFV (ASFV SS-3/Mongolia/2019) detected from a backyard pig in Mongolia in February 2019 using whole genome sequencing. We analyzed their phylogenetic relationship with other genotype II ASFVs from Eurasia. The ASFV SS-3/Mongolia/2019 belonged to genotype II (p72 and p54), serogroup 8 (CD2v), Tet-10a variant (pB602L), and IGRIII variant (intergenic region between the I73R/I329L genes). A total of five amino acid substitutions were observed in MGF 360-10L, MGF 505-4R, MGF 505-9R, NP419L, and I267L genes compared to the ASFV Georgia 2007/1 virus. ML phylogenetic analysis of the whole genome sequence showed that the virus shares a high nucleotide sequence identity with ASFVs recently identified in Eastern Europe and Asia and clustered with the ASFV/Zabaykali/WB5314/2020|Russia|2020 virus which was identified at the border between the Russian Federation and Mongolia in 2020. Our results suggest that trans boundary spread of ASF occurred through close geographic proximity.

Systemic mycosis in a California sea lion (Zalophus californianus) with detection of cystofilobasidiales DNA.

A 6-yr-old, intact male California sea lion (Zalophus californianus) with a systemic mycosis died after 5 wk of antifungal drug therapy. Antemortem clinical findings included hind flipper swelling, ring-lesions on skin of the flippers, and dermal nodules that increased in size and number spreading from the hind flippers and ventral abdomen to the foreflippers and muzzle. Lesions were accompanied by severe lymphadenopathy and development of systemic clinical signs despite therapy using itraconazole and later voriconazole. Histopathologic evaluation of biopsies revealed granulomatous dermatitis due to infection by fungus-producing yeast cells in tissue. Isolation attempts, using biopsied skin and tissue samples collected at necropsy, failed to yield growth of a fungus producing yeast cells like those in histologic section. Consensus polymerase chain reaction (PCR) tests of biopsied skin for fungal DNA produced an amplicon having significant sequence identity with a Cystofilobasidiales, a fungus belonging to a subclade that includes several Cryptococcus spp. Histopathologic evaluation of necropsy tissues revealed a systemic mycosis with yeast cells disseminated throughout subcutis, lymph nodes, and viscera. Hepatic necrosis was identified associated with acute liver failure, possibly from the voriconazole administration. This is the first report documenting the clinical presentation, treatment, and pathologic findings of infection associated with Cystofilobasidiales in a marine mammal and serves to expand the understanding of mycoses in pinnipeds.

Sulawesi tortoise adenovirus-1 in two impressed tortoises (Manouria impressa) and a Burmese star tortoise (Geochelone platynota).

Sulawesi tortoise adenovirus-1 (STAdV-1) is a newly discovered virus infecting endangered and threatened tortoises. It was initially described from a confiscated group of 105 Sulawesi tortoises (Indotestudo forsteni) obtained by the Turtle Survival Alliance and distributed to five sites with available veterinary care across the United States. In a 3-yr period from the initial outbreak, one multi-species collection that rehabilitated and housed adenovirus-infected Sulawesi tortoises experienced deaths in impressed tortoises (Manouria impressa) and a Burmese star tortoise (Geochelone platynota). Impressed tortoises that died had evidence of systemic viral infection with histopathologic features of adenovirus. Adenovirus was identified by consensus nested polymerase chain reaction (PCR) testing and subsequent sequencing of PCR products. Sequencing indicated that the adenovirus infecting these impressed tortoises and Burmese star tortoise was STAdV-1. In one impressed tortoise, viral infection was confirmed using transmission electron microscopy. In situ hybridization using a semiautomated protocol and fluorescein-labeled riboprobe identified STAdV-1 inclusions in spleen, liver, kidney, and testis of one impressed tortoise. The impact of this virus on captive and wild populations of tortoises is unknown; however, these findings indicate that STAdV-1 can be transmitted to and can infect other tortoise species, the impressed tortoise and Burmese star tortoise, when cohabitated with infected Sulawesi tortoises.

Perceptions of pastoralist problems: A participatory study on animal management, disease spectrum and animal health priorities of small ruminant pastoralists in Georgia.

Small ruminants support the livelihoods of millions of poor pastoralist and sedentary households around the world. While pastoralists are generally not amongst the poorest in terms of assets, they are frequently marginalised in terms of their access to political power, health and education. This study was undertaken among pastoralist households keeping small ruminants in four regions of the country of Georgia. Small ruminants are an important cultural, social and economic asset in Georgia and are mainly managed in a transhumant pastoralist system. Georgia suffered its first, and so far only outbreak of peste des petits ruminants (PPR) in 2016. This qualitative interview study was designed to acquire contextual understanding of local small ruminant husbandry and the livelihood situations of the participating pastoralists, and to detect historical, unreported PPR outbreaks. Focus group discussions comprising participatory epidemiology tools and other forms of interviews were used to explore small ruminant management, disease spectrum and management, and animal health priorities. The participants had experienced a wide variety of animal health constraints, with intestinal worms, braxy, piroplasmosis, pasture-related problems, predators and lameness emerging as priorities. No historic, unreported PPR outbreak was detected in this study, and PPR was not a priority for participants. Instead, the day-to-day reality of animal health for the pastoralists was characterised by co-infections of mainly endemic pathogens, and problems related to other challenges such as access to land, feed and genetic resources. The rationale behind the participants' prioritisation of animal health problems was supported by the need to pay extra attention to animals in order to avoid risk factors, keep animals healthy and minimise the negative impact of diseases or management problems; the various epidemiological and clinical parameters of the prioritised diseases; the economic impact of the specific problems and the zoonotic potential of diseases and predation. Even within regions, and within seemingly socially and culturally homogenous groups, there were important local differences in the problems faced by pastoralists that affect their livestock management. This study underlines the importance of a contextualised understanding of the local disease panorama and complexities in the livelihood situations of rural people when designing actions to improve animal health in general or, more specifically, passive surveillance as well as prevention or control measures. Finally, it is concluded that to achieve such an understanding, there is a need for participatory, scoping-style studies that specifically acknowledge diversity and power relations.

Genetic features of Salmonella enterica subspecies diarizonae serovar 61:k:1,5 isolated from abortion cases in sheep, United States, 2020.

Salmonella enterica subspecies diarizonae serovar 61:(k):1, 5, (7) (sheep associated S. diarizonae, SASd) is the most common Salmonella serotype identified in sheep flocks. Despite the involvement with animal and human infections, there is limited information regarding virulence profiles of SASds and their antibiotic resistance gene complement, particularly for those circulating in the U.S. In this study, we genetically characterized three SASds, 20-265, 20-269, and 20-312, isolated from sheep placental tissues during an abortion storm affecting a flock in Connecticut during 2020. SASds were the only bacteria isolated from analyzed sheep tissues. The isolates were sensitive to all the antibiotics tested, but all these SASd isolates carry the aminoglycoside resistance gene, aac(6')-Iaa, and a chromosomal substitution in the parC gene. The proportion of pseudogenes (5.3-5.5%) was similar among the isolates, and these SASds carry IncX1 type plasmids. Comparing with the SASds isolates from Enterobase, the three isolates showed an identical genomic virulence profile carrying virulence genes in the conserved set of other SASd isolates except for steC, iagB, iacP, sseI, and slrP genes. In the SNP-based phylogenetic analysis, SASd sequences were grouped into group A-C, and the group C was further subdivided into subgroup C1-C6. The three isolates clustered with other SASd isolates from the U.S. and Canada in subgroup C6. SASd isolates in the identical phylogenetic groups tended to have similar geographical origin. The results of our study did not provide conclusive evidence about which are the genetic traits that trigger SASds to become virulent in sheep, but our data will provide a point for comparative studies of this Salmonella serovar.

Complete mitochondrial genome of Asian longhorned tick, Haemaphysalis longicornis, Neumann, 1901 (Acari: Ixodida: Ixodidae) identified in the United States.

Haemaphysalis longicornis (Ixodida: Ixodidae), the Asian longhorned tick, which is native to temperate East Asia, has been recently detected in the northeastern region of the United States, drawing concerns about its potential impact on the US animal and public health sectors. Knowledge about the genetic features of H. longicornis found in the US is limited. Therefore, we sequenced the complete mitochondrial genome (mt-genome) from two H. longicornis ticks recently collected in the State of New York, USA, in 2020. These ticks were morphologically identified and tested for tick-borne pathogens at the Connecticut Veterinary Medical Diagnostic Laboratory (Storrs, CT). The mt-genome was 14,694 bp in length and encoded 37 genes, including 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs. Phylogenetic analysis showed that the mt-genome clustered with those of other H. longicornis identified in China. The mt-genome sequence was 99.7% identical to a H. longicornis mt-genome (GenBank: MK439888) collected in China. The cox1 gene haplotype in these ticks belonged to the H1 type, which is the dominant haplotype present in central NJ and Staten Island, NY. The complete mt-genome data are needed to provide insights into genetic changes and phylogenetic studies of H. longicornis ticks.

Genomic Features of Salmonella enterica Subspecies houtenae Serotype 45:g,z51:- Isolated from Multiple Abdominal Abscesses of an African Fat-Tailed Gecko, United States, 2020.

Salmonella enterica subsp. houtenae (S. houtenae) is a common subspecies in reptiles and has been implicated as a source of serious and life-threatening diseases in humans. Although occurrence and significance of S. houtenae infections have been extensively studied, the genetic features of S. houtenae have remained unknown due to a lack of available high-quality genome sequences. We obtained the complete genome sequence of S. houtenae 45:g,z51:- strain 20-369 isolated from multiple abdominal abscesses of an African fat-tailed gecko (Hemitheconyx caudicinctus) using Nanopore and Illumina sequencing technologies and generated the 4.65Mbp complete genome sequence of the S. houtenae str. 20-369. We annotated and analyzed the genome sequence with the aim to gain a deeper understanding of the genome characteristics associated with its pathogenicity. Overall, this study found several interesting genomic features such as pseudogene formation, virulence gene profile, and novel genomic islands. This study provides basis for an understanding possible genetic mechanism underlying pathogenicity of S. houtenae 45:g,z51:- as well as a high-quality genome reference for future comparison studies.

Whole Genome Sequencing and Phylogenetic Analysis of Rabies Viruses from Bats in Connecticut, USA, 2018-2019.

We performed whole genome sequencing and genetic characterization of rabies viruses (RABV) detected in bats submitted to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL) during 2018-2019. Among 88 bats submitted to CVMDL, six brain samples (6.8%, 95% confidence interval: 1.6% to 12.1%) tested positive by direct fluorescent antibody test. RABVs were detected in big brown bats (Eptesicus fuscus, n = 4), a hoary bat (Lasiurus cinereus, n = 1), and an unidentified bat species (n = 1). Complete coding sequences of four out of six detected RABVs were obtained. In phylogenetic analysis, the RABVs (18-62, 18-4347, and 19-2274) from big brown bats belong to the bats EF-E1 clade, clustering with RABVs detected from the same bat species in Pennsylvania and New Jersey. The bat RABV (19-2898) detected from the migratory hoary bat belongs to the bats LC clade, clustering with the eleven viruses detected from the same species in Arizona, Washington, Idaho, and Tennessee. The approach used in this study generated novel data regarding genetic relationships of RABV variants, including their reservoirs, and their spatial origin and it would be useful as reference data for future investigations on RABV in North America. Continued surveillance and genome sequencing of bat RABV would be needed to monitor virus evolution and transmission, and to assess the emergence of genetic mutations that may be relevant for public health.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a Dog in Connecticut in February 2021.

We report the first detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a 3-month-old dog in Connecticut that died suddenly and was submitted to the state veterinary diagnostic laboratory for postmortem examination. Viral RNA was detected in multiple organs of the dog by reverse transcription real time-PCR (RT-qPCR). Negative and positive sense strands of viral RNA were visualized by in situ hybridization using RNAscope technology. Complete genome sequencing and phylogenetic analysis of the hCoV-19/USA/CT-CVMDL-Dog-1/2021 (CT_Dog/2021) virus were conducted to identify the origin and lineage of the virus. The CT_Dog/2021 virus belonged to the GH/B1.2. genetic lineage and was genetically similar to SARS-CoV-2 identified in humans in the U.S. during the winter of 2020-2021. However, it was not related to other SARS-CoV-2 variants identified from companion animals in the U.S. It contained both the D614G in spike and P323L in nsp12 substitutions, which have become the dominant mutations in the United States. The continued sporadic detections of SARS-CoV-2 in companion animals warrant public health concerns about the zoonotic potential of SARS-CoV-2 and enhance our collective understanding of the epidemiology of the virus.