Metabarcoding study to reveal the structural community of strongylid nematodes in domesticated horses in Thailand.

BACKGROUND: Mixed strongylid infections significantly impact equine health and performance. Traditional microscopy-based methods exhibit limitations in accurately identifying strongylid species. Nemabiome deep amplicon sequencing approach previously succeeded in describing the strongylid communities in livestock including equids. However, there are no available studies that describe the structural communities of strongylid parasites in horses in Thailand. Therefore, this study was undertaken encompassing the ITS-2 rDNA metabarcoding assay to characterize strongylid species within horse fecal samples collected from a cohort of yearlings at the largest domesticated stud farm in Thailand. In addition, to investigate the capability of ITS-2 rDNA in assessing the phylogenetic relationships among the identified strongylid species. RESULTS: The study identified 14 strongylid species in the examined equine populations, each with varying prevalence. Notably, Cylicocyclus nassatus and Cylicostephanus longibursatus were identified as the predominant species, with Strongylus spp. conspicuously absent. The phylogenetic analysis of 207 amplicon sequence variants (ASVs) displayed a complex relationship among the investigated cyathostomin species, with some species are positioned across multiple clades, demonstrating close associations with various species and genera. CONCLUSION: The ITS-2 nemabiome sequencing technique provided a detailed picture of horse strongylid parasite species in the studied population. This establishes a foundation for future investigations into the resistance status of these parasites and enables efforts to mitigate their impact.

Zoonotic Vectorborne Pathogens and Ectoparasites of Dogs and Cats in Eastern and Southeast Asia.

To provide data that can be used to inform treatment and prevention strategies for zoonotic pathogens in animal and human populations, we assessed the occurrence of zoonotic pathogens and their vectors on 2,381 client-owned dogs and cats living in metropolitan areas of 8 countries in eastern and Southeast Asia during 2017-2018. Overall exposure to ectoparasites was 42.4% in dogs and 31.3% in cats. Our data cover a wide geographic distribution of several pathogens, including Leishmania infantum and zoonotic species of filariae, and of animals infested with arthropods known to be vectors of zoonotic pathogens. Because dogs and cats share a common environment with humans, they are likely to be key reservoirs of pathogens that infect persons in the same environment. These results will help epidemiologists and policy makers provide tailored recommendations for future surveillance and prevention strategies.

Detection of Ehrlichia canis in canine blood samples by real-time fluorescence resonance energy transfer (FRET) PCR and melting curve analysis.

Ehrlichia canis is a small pleomorphic gram-negative, coccoid, obligatory intracellular bacterium and the cause of canine monocytic ehrlichiosis. A real-time fluorescence resonance energy transfer polymerase chain reaction (real-time FRET PCR) coupled with melting curve analysis was established for detection of E. canis infection in canine blood samples. The VirB9 gene was amplified using one pair of primers and the melting curve analysis was generated by heating the hybridizing probes and amplified products. Eight E. canis-infected dog blood samples were initially identified using the Giemsa staining/microscopic method followed by conventional PCR (cPCR)/Sanger sequencing for confirmation. The sensitivity and specificity of the real-time FRET PCR detection were 87.5% and 100%, respectively and the limit of detection was 6.6 x 10(3) copies of positive E. canis control plasmids. The real-time FRET PCR with melting curve analysis reported here is better than microscopic visualization or cPCR because the method is not affected by the false bias inherent in the microscopic method. Furthermore, many samples can be processed rapidly at the same time. This convenient tool is beneficial as an alternative assay for the epidemiologic study of canine ehrlichiosis as well as for eradication of these organisms in prevention and control programs in endemic areas.

First molecular detection and genetic diversity of Hepatozoon sp. (Apicomplexa) and Brugia sp. (Nematoda) in a crocodile monitor in Nakhon Pathom, Thailand.

The crocodile monitor (Varanus salvator) is the most common monitor lizard in Thailand. Based on habitat and food, they have the potential to transmit zoonoses, with a high possibility of infecting ectoparasites and endoparasites. Diseases that could infect crocodile monitors and be transmitted to other animals, including humans. This research aims to identify and evaluate the phylogenetic relationships of Hepatozoon sp. and sheathed microfilaria in crocodile monitors. The phylogenetic analyses of Hepatozoon, based on 18S rRNA, and sheathed microfilaria, based on the COX1 gene, revealed that the Hepatozoon sp. were grouped with H. caimani, while sheathed microfilaria were grouped together with B. timori. This study provides insights into the genetic diversity and host-parasite interactions of hemoparasites in crocodile monitors in Thailand.

New insight into genetic diversity of zoonotic-potential Ancylostoma ceylanicum in stray cats living in Bangkok, Thailand, based on deep amplicon sequencing.

AIMS: This study aimed to characterize feline hookworms from stray cats living in Bangkok. METHODS AND RESULTS: A total of 56 hookworm-positive faecal samples were identified for hookworm species by using PCR targeting the ITS1, 5.8S, and ITS2 fragment and qPCR targeting ITS2. Of 56 samples, 96.4% (54/56) were identified as Ancylostoma ceylanicum and 1.8% (1/56) as Ancylostoma caninum. With qPCR, 89.3% (50/56) were identified as single A. ceylanicum infection and 5.4% (3/56) as coinfection of A. ceylanicum and A. caninum. For genetic characterization of A. ceylanicum, 10 samples were pooled, and the partial COI gene was amplified, followed by deep amplicon sequencing. Five pooled samples were analysed, and 99.73% were identified with A. ceylanicum sequences, which were allocated into 19 haplotypes (AC01-AC19). Genetic diversity findings for A. ceylanicum in Asia revealed that three of eight haplotypes considered of zoonotic significance occurred in humans, dogs, and cats, including haplotypes H01, H20, and H21. The predominant haplotype in this study, AC01, was clustered with H01-a zoonotic haplotype. CONCLUSIONS: The diversity obtained by deep amplicon sequencing supported that the A. ceylanicum community had high genetic variation. Deep amplicon sequencing was a useful method to determine source, zoonotic potential, and host-parasite relationship.

Unveiling zoonotic threats: molecular identification of Brugia sp. infection in a lion.

Brugia malayi and B. pahangi, potential zoonotic pathogens transmitted by mosquitoes, are believed to primarily infect dogs and cats as reservoir hosts. Although previous studies have indicated nematode infections in lions, particularly in zoo environments where human contact with these reservoirs is possible, limited documentation exists regarding Brugia sp. infections in lions in Thailand. This study aims to investigate a case of Brugia infection in a lion from a zoo in Thailand. The blood sample was collected and examined from a female lion, using staining methods to morphologically identify microfilaria at the genus level. Subsequently, the PCR was employed targeting specific genes, including mitochondrial 12S rDNA, 18S rDNA, cytochrome oxidase I (COI) and Wolbachia surface protein (wsp), to confirm the species of the filarial nematode parasite. The genetic sequencing results revealed a high similarity (99-100%) to B. malayi for the 12S rDNA, 18S rDNA, COI and wsp genes. Phylogenetic analysis based on nucleotide sequences from the 12S rDNA, 18S rDNA, COI and wsp genes showed that the sequences from this study belong to different clusters. This marks the inaugural documentation of molecular identification of Brugia infection in a lion, signifying that lions could function as reservoirs for this parasite and present a potential public health risk in the region. Our research underscores the effectiveness of molecular techniques and phylogenetic analysis in discerning and comprehending the evolution of filarial parasites. Additionally, it emphasizes the significance of these methods in enhancing the diagnosis, control, and prevention of zoonotic filarial nematode infections.

Patterns of Equine Small Strongyle Species Infection after Ivermectin Intervention in Thailand: Egg Reappearance Period and Nemabiome Metabarcoding Approach.

The indiscriminate use of anthelmintics to control parasitic nematodes in horses has led to the emergence of anthelmintic resistance worldwide. However, there are no data available on using ivermectin for treating strongyle infections within domesticated horses in Thailand. Therefore, this study aimed to use the fecal egg count reduction (FECR) test to determine the strongylid egg reappearance period (ERP). Additionally, the nemabiome metabarcoding approach is incorporated to study patterns of strongyle species infection following ivermectin treatment. The study results indicate that, although ivermectin effectively eliminated adult strongyle parasites within two weeks post-treatment, the ERP was shortened to 6 weeks post-treatment with a mean FECR of 70.4% (95% CI 46.1-84.0). This potentially indicates a recent change in drug performance. In addition, nemabiome metabarcoding revealed that strongyle species have different levels of susceptibility in response to anthelmintic drugs. The reduction in ERP was associated with the early reappearance of specific species, dominated by Cylicostephanus longibursatus and Cylicocyclus nassatus, indicating the lower susceptibility of these species. In contrast, Poteriostomum imparidentatum, Triodontophorus nipponicus, and Triodontophorus serratus were not found post-treatment, indicating the high level of susceptibility of these species. This information is vital for comprehending the factors contributing to the emergence of resistance and for devising strategies to manage and control strongyle infections in horses.

Severe fever with thrombocytopenia syndrome (SFTS) in Thailand: using a one health approach to respond to novel zoonosis and its implications in clinical practice.

Severe fever with thrombocytopenia syndrome (SFTS), a tick-borne disease caused by Dabie bandavirus (SFTSV) is an emerging infectious disease of substantial concern in East Asia. In 2019, Ongkittikul S et al. reported the first case of SFTS in Thailand. Our report describes a One Health investigation of SFTS zoonosis examining the index case and suspected animal reservoirs using real-time RT-PCR and immunoassays. We add to the report on the first confirmed case of SFTSV infection in a human in Thailand by conducting a limited but informative One Health surveillance study. Dogs and cats tested positive for SFTSV antibody using IgG ELISA. We conclude that domestic dogs and cats might serve as potential reservoirs for SFTSV spread due to their closer proximity to the index case than other non-domestic animals. Notably, we did not detect SFTSV in synanthropic cats or dogs-nor did we detect SFTSV in Rhipicephalus sanguineus ticks-using RT-PCR. We propose that One Health investigations coupling genomic and serologic assays in response to new SFTS cases could play a pivotal role in preventing and managing SFTS among humans and animals in East Asia. As such, we are establishing a collaborative response to SFTS in Thailand through human outbreak investigations that align with principles of One Health, through environmental surveys and animal RT-PCR and immunoassays. Our investigation highlights the importance of coupling RT-PCR with seroprevalence assays as principal elements of One Health surveillance for SFTS in order to shed light on potential animal reservoirs and track emerging zoonosis.

Successful treatment of Brugia pahangi in naturally infected cats with ivermectin.

Lymphatic filariasis is a common parasitic disease of cats in tropical regions including Thailand. The objective of this study was to determine the efficacy of ivermectin against microfilariae of Brugia pahangi in naturally infected cats. Eight cats naturally infected with B. pahangi were divided into control (untreated) and treated groups. Cats in the latter group were given ivermectin injection at 400 µg/kg weekly for 2 months. Microfilariae were counted every week until 48 weeks. Microfilaremia was significantly decreased in the treated group 4 weeks after starting the treatment and become zero at week 9 and afterwards. On the other hand, cats in the control group had high microfilaremia throughout the study. It was successful to treat and control B. pahangi infection in naturally infected cats using ivermectin.

Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

Pathological effects due to metacercariae of Clinostomum piscidium migration in snakeskin gourami (Trichopodus pectoralis) in Thailand.

Clinostomum spp. is a fish-borne pathogen and a digenetic trematode with a global range. Despite its zoonotic relevance, the pathogenic impact of the parasite in Thai aquaculture is currently unclear. The present study deals with the pathogenic changes that fluke causes in their host, Trichopodus pectoralis, and the molecular confirmation of the Clinostomum piscidium by targeting 18 s rDNA and ITS gene. The metacercariae of C. piscidium were discovered in the body cavity of infected fish. The gross pathological examination revealed a few white migratory tracks on the surface of the liver and spleen. The migratory track showed histologically as a primary hemorrhage and necrosis of hepatic cells surrounded by a layer of macrophages and epithelioid cells, inflammatory cells, and eosinophilic granular cells in the cytoplasm of liver cells and close to the epithelial cells of the intestine. Also, the migratory track in the spleen appeared as a marked decrease of Red Blood Cell (RBC) count and changes in the necrotic tissue. Infection with this metacercaria produced hepatic tissue injury, which disrupted hepatic metabolism and decreased body weight in the fish hosts. The findings of the study suggest that the pathological effect of C. piscidium on farm T. pectoralis can cause significant economic loss by stunting fish development and predisposing fish to opportunistic pathogens in the environment. Hence, the treatment and control of C. piscidium infections are crucial for the viability of the aquaculture sector since this parasite has been found to cause pathological damage to the vital organs of fish.

Genetic diversity and characterization of Wolbachia endosymbiont in canine filariasis.

Canine filariasis is caused by nematodes from the family Onchocercidae, which is transmitted by arthropod vectors. The disease is commonly found in Southeast Asia and exists worldwide. Some filarial nematodes are associated with intracellular bacteria of the genus Wolbachia, which plays an important role in embryogenesis, molting, and the long-term survival of adult worms. This study aims to characterize Wolbachia sp. and determine the association between Wolbachia and canine filarial nematode species in Thailand. A total of 46 dog blood samples that were naturally infected with filarial nematodes were obtained to identify filarial nematode species by Giemsa stained under a light microscope and confirmed using the molecular technique. In order to characterize Wolbachia sp., the nested PCR assay targeting the 16S rRNA gene showed that all samples of Dirofilaria immitis and fifteen samples of Candidatus Dirofilaria hongkongensis were grouped into Wolbachia supergroup C. In addition, all samples of Brugia spp. and five samples of Candidatus Dirofilaria hongkongensis were classified into Wolbachia supergroup D. The genetic diversity analysis conducted using the 16S rRNA gene revealed a similar result when analyzed through phylogenetic tree analysis. This is the first genetic diversity study of Wolbachia of Candidatus Dirofilaria hongkongensis in infected dogs in Thailand.

Molecular identification and genetic diversity of equine ocular setariasis in Thailand based on the COI, 12S rDNA, and ITS1 regions.

Equine ocular setariasis is mainly caused by Setaria digitata, and the identification of this filarial nematode is based on morphology. However, morphological characterization alone is insufficient for the detection and differentiation of S. digitata from its congeners. In Thailand, the molecular detection of S. digitata is lacking and its genetic diversity is still unknown. This study aimed to phylogenetically characterize equine S. digitata from Thailand based on sequences derived from the mitochondrial cytochrome c oxidase subunit 1 (COI), the mitochondrial small subunit ribosomal DNA (12S rDNA), the nuclear internal transcribed spacer 1 (ITS1) and Wolbachia surface protein (wsp). Five samples of S. digitata were characterized, submitted to the NCBI database, and used for phylogenetic analysis as well as the assessment of similarity, entropy, and haplotype diversity. Phylogenetic analyses revealed that the S. digitata Thai strain was similar to S. digitata from China and Sri Lanka, with 99 to 100% similarity. The entropy and haplotype diversity indicated that the S. digitata Thai isolate was conserved and closely related to S. digitata worldwide. This is the first report on the molecular detection of equine ocular setariasis caused by S. digitata in Thailand.

First report of Strongylidae nematode from pilot whale (Globicephala macrorhynchus) by molecular analysis reveals the cosmopolitan distribution of the taxon.

This study investigates the identification, genetic composition, and placement in the evolutionary tree of a particular nematode species found in a short-finned pilot whale in the Gulf of Thailand. To accomplish this, we utilized various methods, including microscopic observations, molecular techniques, and comparative analyses to better understand the characteristics of this parasite. Initially, we concentrated on studying the 18s rDNA sequence through nested PCR, resulting in a 774-bp product. After conducting a BLASTn analysis, we discovered that there were only a few sequences in the GeneBank that shared similarities with our nematode, particularly with Cyathostomum catinatum, although the percent identity was relatively low. To confirm the uniqueness of our sequence, we constructed a phylogenetic tree that demonstrated a distinct branch for our nematode, suggesting significant genetic differentiation from C. catinatum. Additionally, we sequenced a 399-bp section of the ITS2 gene using PCR, and the resulting data showed a close association with the Strongylidae family, specifically with Cylicocyclus insigne. This was further confirmed by BLASTn and CD-HIT-est results, which indicated a 99 and ~94% sequence homology with C. insigne, respectively. The ITS2 phylogenetic tree also supported the position of our isolated sequence within the Strongylidae family, clustering closely with C.insigne. Our findings shed light on the genetic connections, taxonomy, and evolutionary trends within the Strongylidae family, with a particular focus on the widespread nature of the Cylicocyclus genus. This study emphasizes the importance of utilizing molecular techniques and interdisciplinary approaches to gain insight into nematode diversity, evolution, and ecological dynamics in marine environments.

Application of a novel rectangular filtering microfluidic device for microfilarial detection.

The rectangular filtering microfluidic chip was invented using microfluidics device fabrication technology and can separate living microfilariae from blood samples without a syringe pump. The diagnostic results are highly effective. The device is based on the principle of separating millions of blood cells from microfilariae using a rectangular filter structure. It disperses fluid evenly into the flow-passage channel, and its rectangular filter structure is the key to success in reducing the pressure and separating blood cells from microfilariae effectively. The flow rate and blood cell concentration were optimized in our study. The chip is intended to be a point-of-care device that can reduce the use of superfluous instrumentation in the field. The technology is designed to be rapid, accurate, and easy-to-use for all users, especially those in remote areas.

Case report: Thromboembolic heartworm induced lower limb necrosis in a dog.

A 9-year-old spayed female boxer suffered from lameness in both hindlimbs with a perforated paw wound. Additionally, a linear, worm-like creature was penetrating out from the wound. On examination, the dog was emaciated and infected with heartworms, detected through a fresh blood smear, echocardiography, and transabdominal ultrasonography. Adult heartworms were detected at the right atrium (RA), right ventricle (RV), and pulmonary artery (PA), including the distal abdominal aorta, external iliac, and femoral arteries. During the surgery, adults heartworms were removed from both the heart (n = 8) and the femoral arteries (n = 5). Unfortunately, not all heartworms could be removed from these locations due to the extent of the heartworm infection. The opened, ischemic wounds in the distal limbs progressively expanded and the dog subsequently died, possibly due to caval syndrome complications and septicemia. The necropsy showed no evidence of an atrial septal defect, and a total of 25 adult heartworms were collected from the perforated paw, heart, pulmonary, and femoral arteries. All worms collected during the necropsy process were molecularly confirmed to be Dirofilaria immitis.

World Association for the Advancement of Veterinary Parasitology (WAAVP): Second edition of guidelines for evaluating the efficacy of anthelmintics for dogs and cats.

These revised guidelines have been developed to assist in the design, execution, and interpretation of studies to assess the efficacy of anthelmintic drugs against internal parasites in dogs and cats. The design and execution of studies are outlined and discussed. Considerations for specific targeted parasites are included. Information is provided on the principles of selection of animals, procedures for randomization, housing, feeding, necropsy procedures, and record keeping for dose determination dose confirmation studies and field studies. Complementary to the WAAVP general anthelmintic guidelines, these species-specific guidelines should assist investigators in the evaluation of anthelmintic drugs in dogs and cats by using comparable and standardized procedures in studies with appropriate numbers of animals.

Human social conditions predict the risk of exposure to zoonotic parasites in companion animals in East and Southeast Asia.

BACKGROUND: A recent dramatic surge in pet ownership has been observed across metropolitan areas in Asia. To date, there is a dearth of information on the risk associated with pet ownership for the transmission of parasites on a large scale in Asia, despite this continent giving rise to the largest burden of zoonotic infections worldwide. METHODS: We explored the nature and extent of zoonotic internal (endo-) and external (ecto-) parasites and arthropod-borne pathogens in 2381 client-owned dogs and cats living in metropolitan areas of eight countries in East and Southeast Asia using reliable diagnostic tests and then undertook extensive statistical analyses to define predictors of exposure to zoonotic pathogens. RESULTS: The estimated ORs for overall parasite infections are 1.35 [95% CIs 1.07;1.71] in young animals and 4.10 [1.50;11.2] in the animal group older than 15 years as compared with adult animals, 0.61 [0.48;0.77] in neutered animals as compared to unneutered animals, 0.36 [0.26;0.50] in animals living in urban areas as compared with rural areas, 1.14 [1.08;1.21] for each 1 °C increase of annual mean temperature which varies from 12.0 to 28.0 °C, and 0.86 [0.78;0.95] for each year of life expectancy which varies from 70.9 to 83.3 years. CONCLUSIONS: Here we highlight the influence of human life expectancy and the neutering status of the animals, which reflect increased living standards through access to education and human and veterinary health care, to be both strongly associated with exposure to zoonotic parasites. An integrated approach of local and international authorities to implement and manage educational programs will be crucial for the control of zoonotic infections of companion animals in Asia.

Parasites live on or inside animals or humans and can cause disease. Companion animals (pets) with parasites present a potential risk to the health of their owners, as certain kinds of parasites (known as zoonotic parasites) can affect both animal and human health. Here, we investigated whether human social conditions are associated with zoonotic parasite infections in companion animals in East and Southeast Asia. We found that higher human life expectancy and neutering of the companion animals were associated with fewer zoonotic parasite infections in the animals. These findings highlight the need for an enhanced commitment of local authorities to establish prevention campaigns, including education programs, against zoonotic pathogens. These measures will play a crucial role in alleviating the impact of these diseases in companion animals and humans in Asia.

Characterization of serum protein electrophoresis patterns and C-reactive protein in canine tick-borne diseases.

BACKGROUND AND AIM: Canine tick-borne diseases are important diseases with a worldwide distribution. In Thailand, the most important canine tick-borne diseases are ehrlichiosis, babesiosis, and hepatozoonosis. This study aimed to determine the serum protein electrophoresis patterns (SPEPs) and C-reactive protein (CRP) levels associated with Ehrlichia canis , Babesia canis, or Hepatozoon canis single infections. MATERIALS AND METHODS: A total of 650 canine blood samples were collected from animal hospitals and clinics in Bangkok and its vicinity to examine health status and blood parasite infection. Suspected blood parasite infections were examined by buffy coat thin blood smear and confirmed by polymerase chain reaction. Normal dog and positive E. canis , B. canis, and H. canis single infections and serum protein profiles were determined by agarose gel electrophoresis. CRP concentration was measured by fluorescent immunoassay. RESULTS: In dogs infected with E. canis, B. canis, and H. canis single infections, albumin levels and A/G ratios significantly decreased, whereas ß2-globulin levels increased (p<0.05). The γ-globulin level significantly increased in E. canis and H. canis infections (p<0.05). A monoclonal gammopathy pattern wasi observed in E. canis and B. canis sngle infections, whereas ß-γ bridging patterns and increased ß- and γ-globulin fractions were found in H. canis single infections. The CRP level increased in dogs with blood parasite single infections and may be related to the pathogenesis of the infection. CONCLUSION: SPEPs and CRP levels can be used to monitor health status and blood parasite problems in infected dogs.

Emergence of Dirofilaria repens (Spirurida: Onchocercidae) in dogs in Eastern Thailand.

BACKGROUND AND AIM: Dirofilaria repens is a zoonotic vector-borne parasite of dogs and cats. It is not commonly found in every part of Thailand, except the southern part. This study was conducted to investigate the prevalence of this parasite in Eastern Thailand in 2019. MATERIALS AND METHODS: A total of 8003 blood samples were collected from private veterinary clinics and animal hospitals in Eastern Thailand. Blood parasites were examined using buffy coat thin blood smears with Wright-Giemsa staining. D. repens was morphologically identified and confirmed using the acid phosphatase activity technique. RESULTS: The first case of D. repens was found in March 2019. The prevalence of D. repens from January to December 2019 was 0.44% (35/8003) (95% confidence interval 0.30-0.61). CONCLUSION: The prevalence data of D. repens in Eastern Thailand indicate that this parasitic infection should be considered as a zoonotic vector-borne disease. A strategic plan to control zoonotic transmission alongside a preventive program should be emphasized and encouraged among pet owners and veterinarians.