First Reported Cases of SARS-CoV-2 Infection in Companion Animals - New York, March-April 2020.

On April 22, CDC and the U.S. Department of Agriculture (USDA) reported cases of two domestic cats with confirmed infection with SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19). These are the first reported companion animals (including pets and service animals) with SARS-CoV-2 infection in the United States, and among the first findings of SARS-CoV-2 symptomatic companion animals reported worldwide. These feline cases originated from separate households and were epidemiologically linked to suspected or confirmed human COVID-19 cases in their respective households. Notification of presumptive positive animal test results triggered a One Health* investigation by state and federal partners, who determined that no further transmission events to other animals or persons had occurred. Both cats fully recovered. Although there is currently no evidence that animals play a substantial role in spreading COVID-19, CDC advises persons with suspected or confirmed COVID-19 to restrict contact with animals during their illness and to monitor any animals with confirmed SARS-CoV-2 infection and separate them from other persons and animals at home (1).

Clinical and Laboratory Effects of Doxycycline and Prednisolone in Ixodes scapularis-Exposed Dogs With Chronic Anaplasma phagocytophilum Infection.

Persistent infection of Anaplasma phagocytophilum (AP) after treatment and immunosuppression has not been studied in dogs infected with AP after Ixodes scapularis infestation. This descriptive pilot study evaluated 6 laboratory-reared beagles that were persistently positive for AP antibodies after infestation with wild-caught I. scapularis. After 20 weeks, 3 of 6 dogs were administered doxycycline orally for 28 days, and all 6 dogs were then administered prednisolone at 2.2 mg/kg orally for 14 days. Blood was collected from all 6 dogs and evaluated by complete blood count, AP antibodies, and AP DNA at the beginning of the study and on Week 24 through Week 28. Blood was collected from 5 of the dogs on Week 48. No dogs developed recognizable clinical signs of illness or clinically relevant complete blood count abnormalities. During Week 26 through Week 28, all 6 dogs were negative for AP DNA. On Week 48, the 2 doxycycline treated dogs available for testing were negative for AP DNA and antibodies; the 3 untreated dogs were negative for AP DNA but positive for AP antibodies. In this model, the prednisolone protocol used did not activate AP in dogs with chronic, vector-induced infection. Since PCR evidence of AP infection resolved in both groups of dogs, the effect doxycycline had in eliminating AP infection from I. scapularis-exposed dogs will require further study.

Leptospira spp. vaccinal antibodies do not react with Borrelia burgdorferi peptides used in the AccuPlex 4.

We attempted to determine if Leptospira spp. antibodies induced by vaccination would cross-react with Borrelia burgdorferi antigens used in a commercial automated immunofluorescent assay (AccuPlex 4 BioCD; Antech). Staff- and student-owned dogs ( n = 31) were recruited at a veterinary teaching hospital in a B. burgdorferi nonendemic area. The dogs were randomized and administered 1 of 4 commercial Leptospira spp. vaccines that contained serovars Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona, then booster vaccinated 3 wk later. Blood was collected on weeks 0, 3, 4, 8, and 12. After confirming that maximal Leptospira spp. titers occurred on week 4, aliquots of sera from week 4 were shipped frozen for analysis of B. burgdorferi antibodies against OspA, OspC, OspF, P39, and SLP with the AccuPlex system. Week 4 sera from all 31 dogs had a titer of 1:100 for at least 1 Leptospira spp. serovar. Titers of 1:800 or greater were detected against multiple serovars in 27 dogs. None of the samples contained antibodies against the B. burgdorferi OspA, OspC, OspF, P39, and SLP peptides used in the commercial assay. The B. burgdorferi peptides used in the AccuPlex system do not recognize naturally occurring Leptospira spp. antibodies or those induced by the commercial Leptospira spp. vaccines administered in our study.

Genomic profiling of canine mast cell tumors identifies DNA copy number aberrations associated with KIT mutations and high histological grade.

Mast cell tumor (MCT) is the most common skin malignancy of domestic dogs and presents with a widely variable clinical behavior. Although activating KIT mutations are present in approximately 20% of canine MCTs, molecular etiology is largely unknown for the majority of this cancer. Characterization of genomic alterations in canine MCTs may identify genomic regions and/or genes responsible for their development and progression, facilitating the discovery of new therapeutic targets and improved clinical management of this heterogeneous cancer. We performed genome-wide DNA copy number analysis of 109 primary MCTs derived from three popular canine breeds (the Boxer, Labrador Retriever, and Pug) as well as nontarget breeds using oligonucleotide array comparative genomic hybridization (oaCGH). We demonstrated a stepwise accumulation of numerical DNA copy number aberrations (CNAs) as tumor grade increases. DNA sequencing analysis revealed that KIT mutations were found less frequently in the Pug tumors and were strongly associated with high histological grade. Tumors with KIT mutations showed genome-wide aberrant copy number profiles, with frequent CNAs involving genes in the p53 and RB pathways, whereas CNAs were very limited in tumors with wild-type KIT. We evaluated the presence of four CNAs to predict aggressive tumor phenotypes. This approach predicted aggressive tumors with a sensitivity of 78-94% and specificity of 88-93%, when using oaCGH and droplet digital PCR platforms. Further investigation of genome regions identified in this study may lead to the development of a molecular tool for classification and prognosis, as well as identification of therapeutic target molecules.

Anaplasma phagocytophilum infection of domestic cats: 16 cases from the northeastern USA.

OBJECTIVES: Anaplasma phagocytophilum is an Ixodes species-transmitted rickettsial organism that is occasionally associated with clinical abnormalities in humans, ruminants, horses, dogs and cats. While serological evidence of A phagocytophilum exposure is common in cats in Ixodes species endemic areas, reports of clinical feline anaplasmosis are few. The objective of this study was to describe the clinical and laboratory abnormalities and treatment responses in 16 cats with A phagocytophilum DNA amplified from blood. METHODS: Commercial laboratory electronic records were searched to find cats that had A phagocytophilum DNA amplified from their blood. Once cases were identified, the primary care veterinarian was interviewed and the medical records were reviewed. RESULTS: The cats ranged in age from 4 months to 13 years (mean 4.1 years, median 2 years). All cats lived in Ixodes scapularis endemic areas and had potential for exposure. All cats were lethargic, 15 (94%) had elevated body temperature (>39.4°C) and 14 were anorexic on initial physical examination. Other less common clinical findings included hepatosplenomegaly, ataxia, conjunctivitis and elevation of the nictitating membranes. Blood from 11 cats was evaluated by complete blood cell count; abnormalities included lymphopenia in seven (64%) cats, thrombocytopenia in seven (64%), morulae in neutrophils of three (27%), neutropenia in three (27%) and leukopenia in two (18%). Treatment responses were reported for 14 cats, and the clinical abnormalities in these cats resolved when doxycycline was administered. CONCLUSIONS AND RELEVANCE: This is the first published report describing A phagocytophilum morulae in neutrophils of naturally infected North American cats with infection confirmed by PCR. A phagocytophilum infection should be considered in cats evaluated for lethargy, anorexia and fever living in Ixodes species endemic areas.

Multiple antigen target approach using the Accuplex4 BioCD system to detect Borrelia burgdorferi antibodies in experimentally infected and vaccinated dogs.

The primary objective of our study was to optimize detection of serum antibodies to Borrelia burgdorferi using a new commercial automated fluorescence system (Accuplex4 BioCD system, Antech Diagnostics, Lake Success, New York). The system used multiple natural and artificial peptides-outer surface proteins (OspA, OspC, OspF), an outer membrane protein (P39), and a proprietary synthetic peptide (small Lyme peptide [SLP])-and the results were compared with a commercially available enzyme-linked immunosorbent assay that uses a proprietary peptide (C6). Sera from 4 groups were evaluated: dogs vaccinated with 1 of 3 commercially available vaccines (n = 18); dogs infested with adult Ixodes scapularis (black-legged tick; n = 18); dogs previously vaccinated and then infested with I. scapularis (n = 18); and dogs with B. burgdorferi infection that were then vaccinated (n = 14). All of the vaccines evaluated induced OspA responses. However, antibodies against OspF or C6 were not induced in any of the vaccinated dogs. Additionally, the OspF antibodies had 100% sensitivity and specificity when compared to antibodies against C6 peptide. In B. burgdorferi-infected dogs, antibodies against OspC and SLP were detected in serum sooner than antibodies against the other targets. Low levels of antibodies against OspA developed in 6 of 14 B. burgdorferi-infected, unvaccinated dogs and had the shortest duration compared to the other antibodies. Detection of antibody responses to multiple B. burgdorferi targets with this system can be used to help differentiate vaccinated dogs from exposed dogs as well as acute infection from chronic infection.

Comparative cytogenetic characterization of primary canine melanocytic lesions using array CGH and fluorescence in situ hybridization.

Melanocytic lesions originating from the oral mucosa or cutaneous epithelium are common in the general dog population, with up to 100,000 diagnoses each year in the USA. Oral melanoma is the most frequent canine neoplasm of the oral cavity, exhibiting a highly aggressive course. Cutaneous melanocytomas occur frequently, but rarely develop into a malignant form. Despite the differential prognosis, it has been assumed that subtypes of melanocytic lesions represent the same disease. To address the relative paucity of information about their genomic status, molecular cytogenetic analysis was performed on the three recognized subtypes of canine melanocytic lesions. Using array comparative genomic hybridization (aCGH) analysis, highly aberrant distinct copy number status across the tumor genome for both of the malignant melanoma subtypes was revealed. The most frequent aberrations included gain of dog chromosome (CFA) 13 and 17 and loss of CFA 22. Melanocytomas possessed fewer genome wide aberrations, yet showed a recurrent gain of CFA 20q15.3-17. A distinctive copy number profile, evident only in oral melanomas, displayed a sigmoidal pattern of copy number loss followed immediately by a gain, around CFA 30q14. Moreover, when assessed by fluorescence in situ hybridization (FISH), copy number aberrations of targeted genes, such as gain of c-MYC (80 % of cases) and loss of CDKN2A (68 % of cases), were observed. This study suggests that in concordance with what is known for human melanomas, canine melanomas of the oral mucosa and cutaneous epithelium are discrete and initiated by different molecular pathways.

Detection of antibodies against Anaplasma phagocytophilum in dogs using an automated fluorescence based system.

Infection with Anaplasma phagocytophilum can cause significant illness in some dogs and accurate diagnostic assays are needed. The objectives of the study were to optimize an automated fluorescence system for the detection of antibodies against A. phagocytophilum in canine serum. Serum and blood was collected temporally from seven dogs inoculated parenterally with culture-derived A. phagocytophilum and from 36 dogs exposed to wild-caught, adult Ixodes scapularis for 7 days. The system was optimized using the samples from the parenterally inoculated dogs. The ability to detect antibodies against A. phagocytophilum in the I. scapularis exposed dogs by the automated system was compared with a diagnostic kit (ELISA) and an indirect fluorescent antibody assay (IFA). Each blood sample was also assayed for A. phagocytophilum DNA by polymerase chain reaction (PCR). Of the 36 dogs exposed to I. scapularis, A. phagocytophilum DNA was amplified from blood from 22 dogs by PCR with first positive results occurring on weeks 1 (seven dogs), 2 (nine dogs), 3 (four dogs), 4 (one dog), or 5 (one dog). PCR results were positive prior to detection of antibodies in any of the three antibody assays for 19 dogs. The automated fluorescence system and IFA detected antibodies against A. phagocytophilum earlier than the ELISA. In conclusion, A. phagocytophilum PCR assays on blood are indicated in dogs with suspected acute anaplasmosis if serum antibody assays are negative.

Use of an automated system for detection of canine serum antibodies against Ehrlichia canis glycoprotein 36.

Ehrlichia canis is the most common cause of monocytotropic ehrlichiosis in dogs around the world. The purpose of the present study was to validate a new automated fluorescence system (Accuplex4™ BioCD system; Antech Diagnostics, Lake Success, New York) to detect antibodies against the E. canis immunodominant glycoprotein 36 (gp36). Sera and blood samples (ethylenediamine tetra-acetic acid) were collected from mixed sex beagles ( n = 8) on days 0, 3, 7, 10, 14, 17, 21, 28, 42, 49, 56, 63, 70, 77, 84, and 98 after intravenous inoculation with culture-derived E. canis. Sera were assayed using the Accuplex4 BioCD system (Accuplex4), an E. canis indirect fluorescent antibody test (IFAT), and a commercially available kit. A complete blood cell count and a proprietary E. canis polymerase chain reaction (PCR) were performed on each blood sample. On the day thrombocytopenia was first detected for each dog, E. canis DNA was amplified from blood of all dogs. At those times, E. canis antibodies were detected in 7 of 8 dogs by the Accuplex4, 1 of 8 dogs by the commercial kit, and 4 of 8 dogs by IFAT. Ehrlichia canis DNA was amplified from blood before seroconversion in any antibody assay for 6 dogs. Antibodies against gp36 were detected by Accuplex4 within 3 days of PCR-positive test results and were detected up to 25 days sooner than the commercial kit. After starting doxycycline treatment, E. canis DNA was no longer amplified by PCR assay, but serum antibodies remained detectable by all assays.

Prevalence of intestinal parasites in pet dogs in the United States.

To determine the national, regional, and age-related prevalence of intestinal parasites in dogs presenting to veterinarians in the United States, we reviewed the results of examination via zinc sulfate centrifugal flotation of 1,199,293 canine fecal samples submitted to Antech Diagnostics in 2006. The most commonly identified intestinal parasites were ascarids (2.2%), hookworms (2.5%), whipworms (1.2%), Giardia (4.0%), and Cystoisospora (4.4%). With the exception of whipworms, intestinal parasites were more commonly identified in dogs less than 6 months of age (29.6% positive) as compared to those greater than 1 year of age (6.1% positive) although infections with each parasite considered were identified in all age classes of dogs. Hookworm eggs were most commonly identified in fecal samples submitted from dogs from the South (4.0% positive), whereas ascarid eggs and Giardia cysts were most commonly seen in samples from dogs from the West (2.8% and 6.3% positive, respectively). When compared to previous data from shelter dogs, the prevalence of intestinal helminths, particularly ascarids and hookworms, was greatly suppressed in pet dogs in the southern United States (90-91% reduction) and much less so in dogs in the West (52-78% reduction), perhaps due in part to the routine year-round use of monthly anthelmintics effective at controlling both heartworm infection and intestinal helminths in dogs in the South. Taken together these data indicate that intestinal parasites remain a common, important finding in dogs presenting to veterinary practices although in most of the country infection rates in pet dogs appear to be greatly reduced from the level reported from dogs in animal shelters.