A De Novo Whole Genome Assembly and Annotation of Parelaphostrongylus tenuis.

Parelaphostrongylus tenuis causes ungulate morbidity and mortality in eastern and central North America, but no reference genome sequence exists to facilitate research. Here, we present a P. tenuis genome assembly and annotation, generated with PacBio and Illumina technologies. The assembly is 491 Mbp, with 7285 scaffolds and 185 kb N50.

Possible Cross-Reactivity of Feline and White-Tailed Deer Antibodies against the SARS-CoV-2 Receptor Binding Domain.

In late 2019, a novel coronavirus began circulating within humans in central China. It was designated SARS-CoV-2 because of its genetic similarities to the 2003 SARS coronavirus (SARS-CoV). Now that SARS-CoV-2 has spread worldwide, there is a risk of it establishing new animal reservoirs and recombination with native circulating coronaviruses. To screen local animal populations in the United States for exposure to SARS-like coronaviruses, we developed a serological assay using the receptor binding domain (RBD) from SARS-CoV-2. SARS-CoV-2's RBD is antigenically distinct from common human and animal coronaviruses, allowing us to identify animals previously infected with SARS-CoV or SARS-CoV-2. Using an indirect enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2's RBD, we screened serum from wild and domestic animals for the presence of antibodies against SARS-CoV-2's RBD. Surprisingly prepandemic feline serum samples submitted to the University of Tennessee Veterinary Hospital were ∼50% positive for anti-SARS RBD antibodies. Some of these samples were serologically negative for feline coronavirus (FCoV), raising the question of the etiological agent generating anti-SARS-CoV-2 RBD cross-reactivity. We also identified several white-tailed deer from South Carolina with anti-SARS-CoV-2 antibodies. These results are intriguing, as cross-reactive antibodies toward SARS-CoV-2 RBD have not been reported to date. The etiological agent responsible for seropositivity was not readily apparent, but finding seropositive cats prior to the current SARS-CoV-2 pandemic highlights our lack of information about circulating coronaviruses in other species. IMPORTANCE We report cross-reactive antibodies from prepandemic cats and postpandemic South Carolina white-tailed deer that are specific for that SARS-CoV RBD. There are several potential explanations for this cross-reactivity, each with important implications to coronavirus disease surveillance. Perhaps the most intriguing possibility is the existence and transmission of an etiological agent (such as another coronavirus) with similarity to SARS-CoV-2's RBD region. However, we lack conclusive evidence of prepandemic transmission of a SARS-like virus. Our findings provide impetus for the adoption of a One Health Initiative focusing on infectious disease surveillance of multiple animal species to predict the next zoonotic transmission to humans and future pandemics.

Patterns of antimicrobial, multidrug and methicillin resistance among Staphylococcus spp. isolated from canine specimens submitted to a diagnostic laboratory in Tennessee, USA: a descriptive study.

BACKGROUND: Multidrug- and methicillin-resistant staphylococci are both veterinary and public health concerns due to their zoonotic potential. Therefore, the objective of this study was to investigate patterns of antimicrobial, multidrug, and methicillin resistance among four Staphylococcus spp. commonly isolated from canine clinical specimens submitted to the Clinical Bacteriology Laboratory at the University of Tennessee College of Veterinary Medicine (UTCVM). METHODS: Results of antimicrobial susceptibility testing and mecA polymerase chain reaction (PCR) for isolates of four common Staphylococcus spp. isolates were obtained from the Bacteriology Laboratory at the UTCVM between 01/01/2006 and 12/31/2017. Cochran-Armitage trend test was used to assess temporal trends of antimicrobial resistance (AMR), multidrug resistance (MDR), and methicillin resistance. Kappa test of agreement was used to assess agreement between the results of PCR and disk diffusion tests. RESULTS: Most of the 7805 isolates were S. pseudintermedius (6453 isolates), followed by S. coagulans (860), S. aureus (330), and S. schleiferi (162). Among S. pseudintermedius isolates, 45.5% were MDR, and 30.8% were methicillin-resistant (MRSP). There was a significant temporal increase in MRSP (p = 0.017). Chloramphenicol resistance increased among both MRSP and methicillin-susceptible (MSSP) isolates (p <  0.0001). Among S. aureus isolates, 40.9% were MDR, 37.4% were methicillin-resistant (MRSA), and the proportion of MRSA isolates increased significantly (p = 0.0480) over time. There was an increasing temporal trend in the proportion of MDR isolates among MSSP (p = 0.0022), but a decrease among MRSP (p <  0.0001) and MRSA (p = 0.0298). S. schleiferi had the highest percentage (56.9%) of methicillin-resistant isolates. Oxacillin disk diffusion was superior to cefoxitin for the detection of mecA-mediated resistance and had almost perfect agreement with mecA PCR assay for S. pseudintermedius (95.4% agreement, kappa (κ) = 0.904; p <  0.0001), S. coagulans (95.6%, κ = 0.913; p <  0.0001) and S. schleiferi (97.7%, κ = 0.945; p <  0.0001). However, cefoxitin disk diffusion was superior to oxacillin disk diffusion and had almost perfect agreement with mecA PCR assay for S. aureus (95.3%, κ = 0.834; p <  0.0001). CONCLUSIONS: The levels of resistance and increasing temporal trends are concerning. These findings have implications for treatment decisions and public health due to the zoonotic potential of staphylococci. Continued surveillance and use of antibiograms to guide clinical decisions will be critical.

Disseminated Mycobacterium genavense infection in a guinea pig (Cavia porcellus): a case report.

BACKGROUND: Mycobacteria are found in many environmental conditions and infect a variety of species, including rodents and rabbits. Guinea pigs are used experimentally as a model for Mycobacterium tuberculosis, but natural mycobacteriosis in guinea pigs has not been reported. CASE PRESENTATION: A 1.5-year-old female guinea pig was found acutely deceased with no premonitory illness. On gross post-mortem examination, multifocal to coalescing, raised, firm, pale tan nodules with discrete, irregular margins were noted over the surfaces of all lung lobes. Histopathology revealed nodules composed of clustered foamy macrophages and multinucleated giant cells containing numerous bacterial rods. Similar bacteria-laden macrophages were noted within sections of the liver, heart, palpebral conjunctiva, duodenum, and cecum. Polymerase chain reaction was performed on tissues collected during post-mortem examination. The 16S rRNA gene product was sequenced and was identical to the Mycobacterium genavense type strain. CONCLUSIONS: To the best of the author's knowledge, this report details the first documented case of Mycobacterium genvaense infection in a guinea pig and a follow up investigation of close-contact animals. Given their experimental susceptibility and this clinical case report, mycobacteriosis should be considered as a differential in guinea pigs exhibiting weight loss in the absence of other clinical signs. With the potential for zoonotic transmission in immunosuppressed individuals, precautions should be taken to safeguard human health in cases of guinea pigs with suspected M. genavense infection.

Macrophage-Mediated Phagocytosis and Dissolution of Amyloid-Like Fibrils in Mice, Monitored by Optical Imaging.

Light chain-associated amyloidosis is characterized by the extracellular deposition of amyloid fibrils in abdominothoracic organs, skin, soft tissue, and peripheral nerves. Phagocytic cells of the innate immune system appear to be ineffective at clearing the material; however, human light chain amyloid extract, injected subcutaneously into mice, is rapidly cleared in a process that requires neutrophil activity. To better elucidate the phagocytosis of light chain fibrils, a potential method of cell-mediated dissolution, amyloid-like fibrils were labeled with the pH-sensitive dye pHrodo red and a near infrared fluorophore. After injecting this material subcutaneously in mice, optical imaging was used to quantitatively monitor phagocytosis and dissolution of fibrils concurrently. Histologic evaluation of the residual fibril masses revealed the presence of CD68+, F4/80+, ionized calcium binding adaptor molecule 1- macrophages containing Congo red-stained fibrils as well as neutrophil-associated proteins with no evidence of intact neutrophils. These data suggest an early infiltration of neutrophils, followed by extensive phagocytosis of the light chain fibrils by macrophages, leading to dissolution of the mass. Optical imaging of this novel murine model, coupled with histologic evaluation, can be used to study the cellular mechanisms underlying dissolution of synthetic amyloid-like fibrils and human amyloid extracts. In addition, it may serve as a test bed to evaluate investigational opsonizing agents that might serve as therapeutic agents for light chain-associated amyloidosis.

Staphylococcus ursi sp. nov., a new member of the 'Staphylococcus intermedius group' isolated from healthy black bears.

Six Staphylococcus strains were isolated from healthy black bears (Ursus americanus) in the Great Smoky Mountains National Park, Tennessee, USA. Phylogenetic analysis based on complete genome, 16S rRNA, dnaJ, hsp60, rpoB and sodA genes, and MALDI-TOF-MS main spectral profiles revealed that the strains belonged to one species and showed the closest relatedness to members of the 'Staphylococcus intermedius group' (SIG), which include Staphylococcus intermedius, Staphylococcus pseudintermedius, Staphylococcus delphini and Staphyloccoccus cornubiensis. The strains were positive in SIG-specific and negative in individual species-specific PCR assays for the nuc gene. The strains can be differentiated from the other SIG species by the absence of sucrose fermentation, from S. intermedius DSM 20373T, S. pseudintermedius CCUG 49543T and S. cornubiensis DSM 105366T by the absence of methyl ß-d-glucopyranoside fermentation and from S. delphini DSM 20771T by fermentation of trehalose. DNA relatedness of the type strain MI 10-1553T with the type strains of S. delphini, S. pseudintermedius, S. intermedius and S. cornubiensis was ≤48.2 % by digital DNA-DNA hybridization and ≤92.3 % by average nucleotide identity calculations. Iso-C15:0, anteiso-C15 : 0 and anteiso-C17 : 0 were the most common fatty acids. Polar lipids consisted of phosphadidylglycerols, phospholipids, glycolipid, diphosphatidylglycerol and aminophospholipid. Cell-wall peptidoglycan was of type A3α l-Lys-Gly3 (Ser; similar to A11.2 and A11.3). The respiratory quinone belonged to menaquinone 7 (MK-7). The G+C content of MI 10-1553T was 39.3 mol%. The isolated strains represent a novel species of the genus Staphylococcus, for which we propose the name Staphylococcus ursi sp. nov. The type strain is MI 10-1553T (=ATCC TSD-55T=CCOS 1900T).

Identification, Cloning, and Characterization of Staphylococcus pseudintermedius Coagulase.

Coagulase activation of prothrombin by staphylococcus induces the formation of fibrin deposition that facilitates the establishment of infection by Staphylococcus species. Coagulase activity is a key characteristic of Staphylococcus pseudintermedius; however, no coagulase gene or associated protein has been studied to characterize this activity. We report a recombinant protein sharing 40% similarity to Staphylococcus aureus coagulase produced from a putative S. pseudintermedius coagulase gene. Prothrombin activation by the protein was measured with a chromogenic assay using thrombin tripeptide substrate. Stronger interaction with bovine prothrombin than with human prothrombin was observed. The S. pseudintermedius coagulase protein also bound complement C3 and immunoglobulin. Recombinant coagulase facilitated the escape of S. pseudintermedius from phagocytosis, presumably by forming a bridge between opsonizing antibody, complement, and fibrinogen. Evidence from this work suggests that S. pseudintermedius coagulase has multifunctional properties that contribute to immune evasion that likely plays an important role in virulence.

The prevalence of Dermatophilus congolensis in horses with pastern dermatitis using PCR to diagnose infection in a population of horses in southern USA.

BACKGROUND: Dermatophilus congolensis is a facultative anaerobic actinomycete that causes papular to exudative dermatitis with crusting in horses. This organism is frequently implicated as a cause of pastern dermatitis, but few data are available validating the organism's association with this disease. HYPOTHESIS/OBJECTIVES: The aim of this study was to evaluate if D. congolensis is associated with pastern dermatitis in horses utilizing RT-qPCR. ANIMALS: Fifteen client-owned horses diagnosed with pastern dermatitis and eight client-owned unaffected control horses were utilized for this study. METHODS: A cross-sectional study was performed. History and physical examination findings were recorded, and samples were collected and tested for D. congolensis utilizing cytological evaluation and RT-qPCR. Dermatophyte culture and superficial skin scrapings were also performed. RESULTS: Ten of 15 horses with pastern dermatitis had feathered pasterns. Dermatophilus congolensis was identified by RT-qPCR from one nonfeathered horse but none with feathered pasterns. Cytological evaluation identified bacteria in all horses but failed to identify organisms resembling D. congolensis in any horse. Four of 15 horses, all feathered, were positive for Chorioptes mites. Fungal culture was negative for dermatophytes in all horses. All test results were negative for the eight control horses. CONCLUSIONS AND CLINICAL IMPORTANCE: Dermatophilus congolensis was uncommonly associated with pastern dermatitis in horses in this population. However, chorioptic mange was commonly associated with pastern dermatitis in feathered horses and represented an important differential diagnosis for this clinical presentation.

PCR analysis of a prescription vegetarian diet and use in three dogs with cutaneous adverse food reactions.

BACKGROUND: Cutaneous adverse food reaction (CAFR) is diagnosed by performing an elimination diet trial utilizing prescription or home-cooked diets followed by provocative challenge. OBJECTIVES: To report findings of PCR analysis of a prescription vegetarian diet (RCV) for undeclared proteins of animal origin, as well as to describe its utilization for diagnosis and management of dogs suspected of having CAFR. ANIMALS: Three client-owned dogs. METHODS: PCR analysis of RCV for 11 mammalian species and poultry. In three dogs, clinical examination, cytology, aerobic culture (if indicated) and at least one elimination diet trial with RCV. RESULTS: In our case series, all dogs had a history of pruritus and recurrent pyoderma that resolved with infection control and an elimination diet trial. In cases 1 and 2, a diagnosis of CAFR was made following an elimination trial with RCV and provocative challenge. Case 3 had a previously confirmed diagnosis of CAFR and RCV was successfully used to maintain remission of CAFR-related signs. PCR testing of RCV was negative for 11 mammalian species and poultry. CONCLUSIONS AND CLINICAL IMPORTANCE: The RCV diet was found not to contain any undeclared mammalian or avian proteins. In this case series, the RCV was successfully used to diagnose and maintain three dogs with CAFR.

New Macrolide-Lincosamide-Streptogramin B Resistance Gene erm(48) on the Novel Plasmid pJW2311 in Staphylococcus xylosus.

Whole-genome sequencing of Staphylococcus xylosus strain JW2311 from bovine mastitis milk identified the novel 49.3-kb macrolide-lincosamide-streptogramin B (MLSB) resistance plasmid pJW2311. It contained the macrolide resistance gene mph(C), the macrolide-streptogramin B resistance gene msr(A), and the new MLSB resistance gene erm(48) and could be transformed into Staphylococcus aureus by electroporation. Functionality of erm(48) was demonstrated by cloning and expression in S. aureus.

Evaluation of Pyrrolidonyl Arylamidase Activity in Staphylococcus delphini.

Clinical reference textbooks lack data for pyrrolidonyl arylamidase (PYR) activity in Staphylococcus delphini This study evaluated PYR activities of 21 S. delphini strains by reference broth, rapid disc, and rapid slide methods. Species and subgroup identifications were confirmed by nucleic acid-based methods and included nine group A and 12 group B strains. Testing by rapid PYR methods with products from four manufacturers was performed at two testing locations, and, with the exception of one strain tested at one location using reagents from one manufacturer, each S. delphini strain tested positive for PYR activity. Therefore, PYR may be a useful single-test adjunct for distinguishing Staphylococcus aureus from S. delphini and other members of the Staphylococcus intermedius group.

RT-qPCR for the diagnosis of dermatophilosis in horses.

BACKGROUND: Dermatophilus congolensis causes a crusting dermatitis that affects horses. Diagnosis requires the identification of the organism with cytological evaluation of crust samples. This method can lack sensitivity in chronic cases. HYPOTHESIS/OBJECTIVES: To develop a probe-based real time quantitative polymerase chain reaction (RT-qPCR) test to assist with the diagnosis of dermatophilosis in horses. ANIMALS: Twenty six privately owned horses and seven horses from a research colony were used. METHODS: Crust samples, collected from 14 horses with suspected dermatophilosis and 12 horses with crusting skin disease not characteristic of dermatophilosis, were evaluated by cytological evaluation and RT-qPCR; the latter was also performed on hair samples collected from seven healthy horses. RESULTS: Cytological evaluation revealed organisms consistent with Dermatophilus congolensis from nine horses with suspected dermatophilosis, with only a few organisms seen from five samples. Cytological evaluation of all other crusts was negative for Dermatophilus. Other bacterial organisms were detected on cytological evaluation from 15 samples. RT-qPCR for Dermatophilus was positive from 11 crusts, whereas all other samples were negative. Two samples were cytologically negative but RT-qPCR positive for Dermatophilus. No samples were cytologically positive but RT-qPCR negative for Dermatophilus. CONCLUSION: Results of this study show that RT-qPCR may be a more sensitive and easier method than cytological evaluation for the diagnosis of dermatophilosis in horses.

Dorsal black skin necrosis in a Vietnamese pot-bellied pig.

BACKGROUND: Thermal burns are an uncommon cause of injury in large animals. CASE REPORT: A 10-month-old pet female black and white Vietnamese pot-bellied pig presented to the emergency service with fever and erythematous to purpuric skin lesions affecting the intermandibular space and hocks. One week prior to the emergency visit, she had appeared restless and in pain. Two weeks following the emergency visit, she again presented to the large animal clinic with sloughing of the pigmented skin on her head, face, dorsal and lateral trunk sparing the nonpigmented skin and pigmented ears. The affected skin constituted ~40% of her total skin. Histopathological findings for affected skin included full-thickness epidermal and partial to full-thickness dermal coagulative necrosis with follicular epithelial mineralization, while that from normal-appearing pigmented skin was within normal limits. A culture from a skin biopsy yielded meticillin-resistant Staphylococcus aureus (ST-72). Treatments included oral antibiotics, pain management, hyperbaric oxygen therapy and general anaesthesia to facilitate debridement. Healthy skin was often present when the necrotic skin was debrided, although in some areas the necrosis extended into the underlying fat. Complications that occurred during rehabilitation included intense pruritus that resulted in self-trauma and the formation of a nasal fistula, which was later surgically corrected. CONCLUSIONS AND CLINICAL IMPORTANCE: Cases of dorsal thermal necrosis in pot-bellied pigs are uncommon in the literature. Based on the clinical presentation and lack of another identifying cause, the lesions were attributed to a sun-induced thermal burn.

PCR amplification and DNA sequence identification of an unusual morphological form of Demodex cati in a cat.

BACKGROUND: Molecular characterization of Demodex mites is being used to identify mite species in dogs. This technique is now being applied to cat Demodex species, allowing for better characterization of the mites. HYPOTHESIS/OBJECTIVES: Molecular diagnostics will clarify the existence of diverse Demodex mites identified morphologically. ANIMALS: A cat with generalized demodicosis secondary to chronic steroid treatment for erythroid dysplasia. METHODS: Skin scrapings demonstrated large numbers of follicular mites consistent with Demodex cati as well as a morphologically different Demodex mite with a blunted abdomen. The 16S rRNA DNA was amplified by PCR, sequenced and compared with available Demodex sequences, including Demodex cati, Demodex gatoi and an unnamed Demodex sp. RESULTS: A single PCR product was obtained, the DNA sequence of which was an exact match with D. cati. CONCLUSIONS AND CLINICAL IMPORTANCE: The shorter unnamed mite was not a different species in this case, but a different morphological form of D. cati. This report demonstrates the utility of molecular diagnostics to clarify the identity of mites that differ morphologically.

Whole Genome Sequencing and Comparative Genomics of Six Staphylococcus schleiferi and Staphylococcus coagulans Isolates.

Staphylococcus schleiferi and Staphylococcus coagulans, closely related bacterial species within the Staphylococcus genus, present a challenge in classification and diagnosis due to their close genetic proximity and overlapping phenotypic features. Moreover, our understanding of the virulence mechanisms in staphylococcal species, beyond the extensively studied Staphylococcus aureus, remains limited, underscoring the importance of using comparative data to enhance our insights into virulence within these bacterial species. This study employed a comprehensive approach, utilizing comparative genomics, to identify genomic distinctions between S. schleiferi and S. coagulans, aiming to address the challenges in the accurate classification and diagnosis of these organisms and identify unique features. Whole genome sequencing was performed on six clinical isolates, and their genomes were compared to identify variations in gene content and virulence factors. De novo assembly and annotation revealed two samples as S. coagulans and four samples as S. schleiferi. Analysis of the core genomes revealed conserved regions crucial for defining species identity, while accessory genomic elements contained unique genes, possibly impacting the pathogenicity of the species.

Distinguishing characteristics of Staphylococcus schleiferi and Staphylococcus coagulans of human and canine origin.

Staphylococcus schleiferi and Staphylococcus coagulans are opportunistic pathogens of animals and humans. They were previously classified as Staphylococcus schleiferi subs. schleiferi and Staphylococcus schleiferi subs. coagulans, respectively, and recently reclassified as separate species. S. coagulans, is frequently associated with dogs, whereas S. schleiferi is more commonly isolated from humans. Coagulase activity status is a defining characteristic of the otherwise closely related species. However, the use of coagulase tests originally developed to distinguish S. aureus from non-coagulase-producing staphylococci, for this purpose is questionable and the basis for their host preference has not been elucidated. In the current study, a putative coa gene was identified and correlated with coagulase activity measured using a chromogenic assay with human and bovine prothrombin (closely related to canine prothrombin). The results of the tests performed with human prothrombin showed greater reactivity of S. coagulans isolates from humans than isolates obtained from dogs with the same substrate. Our data suggest that unlike S. coagulans isolates from humans, isolates from dogs have more coagulase activity with bovine prothrombin (similar to canine prothrombin) than human prothrombin. Differences in nuc and 16s rRNA genes suggest a divergence in S. coagulans and S. schleiferi. Phenotypic and genotypic variation based on the number of IgG binding domains, and the numbers of tandem repeats in C-terminal fibronectin binding motifs was also found in protein A, and fibronectin-binding protein B respectively. This study identified a coa gene and associated phenotypic activity that differentiates S. coagulans and S. schleiferi and identified key phylogenetic and phenotypic differences between the species.

PCR amplification and DNA sequencing of Demodex injai from otic secretions of a dog.

BACKGROUND: The identification of Demodex mites from dogs is usually based on morphology and location. Mites with uncharacteristic features or from unusual locations, hosts or disease manifestations could represent new species not previously described; however, this is difficult to determine based on morphology alone. HYPOTHESIS/OBJECTIVES: The goal of this study was to identify and confirm Demodex injai in association with otitis externa in a dog using PCR amplification and DNA sequencing. METHODS: Otic samples were obtained from a beagle in which a long-bodied Demodex mite was identified. For comparison, Demodex mite samples were collected from a swab and scraping of the dorsal skin of a wire-haired fox terrier and an otic sample from a dog with generalized and otic demodicosis. To identify the Demodex mite, DNA was extracted, and 16S rRNA was amplified by PCR, sequenced and compared with Demodex sequences available in public databases and from separate samples morphologically diagnosed as D. injai and Demodex canis. RESULTS: PCR amplification of the long-bodied mite rRNA DNA obtained from otic samples was approximately 330 bp and was identical to that from the mite morphologically identified as D. injai obtained from the dorsal skin of a dog. Furthermore, the examined mite did not have any significant homology to any of the reported genes from Demodex spp. CONCLUSIONS: These results confirmed that the demodex mites in this case were D. injai.

A molecular technique for the detection and differentiation of Demodex mites on cats.

BACKGROUND: Demodex gatoi causes a pruritic dermatitis in cats. Diagnosis requires the demonstration of mites using superficial skin scrapings or faecal flotation, which can be insensitive. HYPOTHESIS/OBJECTIVES: The goal of this study was to develop a molecular method to diagnose D. gatoi infection in cats and distinguish these mites from Demodex cati. ANIMALS: Fifty-three shelter cats, 11 cats from a closed research colony and 12 privately owned cats were used. METHODS: Demodex gatoi and D. cati were obtained from scrapings of cat skin. The 16S rRNA DNA was amplified by PCR, sequenced and compared with available Demodex sequences. Hair and skin samples were also collected for microscopic examination and DNA isolation. RESULTS: DNA sequences were obtained from D. gatoi and D. cati. qPCR with D. gatoi specific primers and probe amplified DNA isolated from D. gatoi and not D. cati. Conversely, D. cati qPCR primers and probe amplified D. cati DNA and not D. gatoi. Five of the shelter cats were positive for D. gatoi. Two of these cats were pruritic, and the other three were in contact with these cats. Only one cat was positive for D. gatoi on skin scraping but was negative for D. gatoi or D. cati DNA. CONCLUSION: Results from this study show D. gatoi and D. cati to be distinct species. A novel qPCR test for the identification and differentiation of D. gatoi and D. cati was developed. Once optimized, this test could provide a valuable technique for the diagnosis of D. gatoi infection.