Occurrence and Risk Factors Associated with Platynosomum illiciens Infection in Cats with Elevated Liver Enzymes.

Platynosomum spp., a hepatic trematode, causes fatal hepatobiliary disease in cats. Feline platynosomiasis is often underestimated due to a lack of awareness and diagnostic challenges. This study aimed to investigate the occurrence, factors, and clinicopathological abnormalities associated with Platynosomum spp. infection in cats with elevated serum ALT levels. Platynosomum infection was determined using zinc sulfate flotation and formalin-ether sedimentation. DNA sequence analysis of PCR products from the Platynosomum internal transcribed spacer 2 (ITS2) region and cox1 gene was used to identify Platynosomum species. Of a total of 43 cat fecal samples, the proportion of Platynosomum spp. infection by microscopic examination was 11.63% (5/43). All PCR-positive samples were molecularly identified as Platynosomum illiciens. From the logistic regression analysis, the odds of Platynosomum infection in cats without a deworming program were 16 times higher than those of regularly dewormed cats. Demographic data, housing conditions, and predatory behavior were not significantly associated with the infection. Regarding blood profiles, infected cats had higher eosinophil counts (p = 0.014), with no significant differences in ALT (p = 0.791) or ALP (p = 0.970) levels compared to non-infected cats. Our findings demonstrate that eosinophilia in cats with increased serum ALT may suggest P. illiciens infection in endemic areas. We strongly recommend a regular deworming program to mitigate the risk of P. illiciens infection.

High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus.

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 101 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

First study on the immunohistochemical expression of cyclooxygenase-2 and clinicopathological association in canine hepatoid gland neoplasms.

Background and Aim: Hepatoid gland neoplasms (HGNs) constitute one of the most common cutaneous tumors that arise from perianal glands in dogs and are clinically characterized by rapid growth. Cyclooxygenase-2 (COX-2), the inducible form of the enzyme, is associated with several hallmarks of tumorigenesis. Its expression has been confirmed in several human and animal neoplastic tissues, but there are no reports in hepatoid gland tissues. Therefore, this study aimed to investigate COX-2 immunoexpression in canine HGNs, compare the expression among groups of normal hepatoid glands, hepatoid gland adenomas (HGAs), hepatoid gland epitheliomas (HGEs), and hepatoid gland carcinomas (HGCs), and assess the association of the COX-2 expression with clinicopathological features. Materials and Methods: Sixty-one formalin-fixed paraffin-embedded canine hepatoid gland tissues (20 samples of HGAs, 16 of HGEs, 15 of HGCs, and 10 of normal hepatoid glands) were analyzed for COX-2 expression using immunohistochemistry with scoring for percentage positivity and intensity. Multiple comparisons of COX-2 expression among normal and neoplastic hepatoid glands and the associations between COX-2 expression and clinicopathological features were analyzed. Results: Cyclooxygenase-2 expression was not detected in 60% of normal hepatoid glands and 25% of HGAs. Seventy-five percent of HGAs had a weak expression, while 43.7% and 56.3% of HGEs showed weak and moderate expression, respectively. The expression of HGCs ranged from weak (13.3%) to moderate (33.3%) and strong (53.3%). The immunoreactivity score of COX-2 labeling was significantly different among the normal and neoplastic hepatoid glands (p < 0.0001). The highest score was observed in the HGCs. Only in HGCs, the strong COX-2 expression was significantly associated with some clinicopathological features, including tissue invasion (p = 0.007) and necrosis (p = 0.029). Conclusion: These results suggest that COX-2 may play a role in the modulation of neoplastic cell growth. These preliminary data lead to further investigation on the potential of COX-2 expression as a prognostic indicator and COX-2 inhibitors for canine HGCs treatment.

Recombinant Ehrlichia canis GP19 Protein as a Promising Vaccine Prototype Providing a Protective Immune Response in a Mouse Model.

The intracellular bacterium Ehrlichia canis is the causative pathogen of canine monocytic ehrlichiosis (CME) in dogs. Despite its veterinary and medical importance, there is currently no available vaccine against this pathogen. In this study, the recombinant GP19 (rGP19) was produced and used as a recombinant vaccine prototype in a mouse model against experimental E. canis infection. The efficacy of the rGP19 vaccine prototype in the part of stimulating B and T cell responses and conferring protection in mice later challenged with E. canis pathogen were evaluated. The rGP19-specific antibody response was evaluated by ELISA after E. canis challenge exposure (on days 0, 7, and 14 post-challenge), and demonstrated significantly higher mean antibody levels in rGP19-immunized mice compared with adjuvant-immunized and naive mice. Significantly lower ehrlichial loads in blood, liver, and spleen DNA samples were detected in the immunized mice with rGP19 by qPCR. The up-regulation of IFNG and IL1 mRNA expression were observed in mice immunized with rGP19. In addition, this study detected IFN-γ-producing memory CD4+ T cells in the rGP19-immunized mice and later infected with E. canis on day 14 post-infection period using flow cytometry. The present study provided a piece of evidence that rGP19 may eliminate E. canis by manipulating Th1 and B cell roles and demonstrated a promising strategy in vaccine development against E. canis infection in the definitive host for further study.

Effect of GP19 Peptide Hyperimmune Antiserum on Activated Macrophage during Ehrlichia canis Infection in Canine Macrophage-like Cells.

In terms of its veterinary importance, vaccine development against Ehrlichia canis is needed. However, the effect of developing vaccines on humoral immune response against E. canis infection is still unknown. Novel GP194-43 was synthesized according to E. canis GP19 epitope prediction. To restrict any loss and/or illness in the host animal, rabbits were used in this study to produce GP194-43 hyperimmune sera. The effect of GP194-43 hyperimmune sera on neutralization was examined in vitro by determining the inhibition of E. canis infection of the macrophage-like cell line (DH82) in the presence of the sera. Four groups of DH82 cells received differing treatments. These included E. canis experimentally infected DH82 cells, E. canis-infected DH82 cells with control rabbit serum (untreated group), E. canis-infected DH82 cells with GP194-43 rabbit antiserum (treated group) and uninfected cells (negative control group), respectively. The treated group developed a decrease (p < 0.01) in the percentage of E. canis infected cells after 3 days post-infection at 48.57 ± 1.28. In addition, real-time PCR analyses of cytokine mRNA expression involved with the macrophage, humoral, and cellular immune responses were conducted. The findings revealed an upregulated expression of IFNG in the treated group during the infection. This study demonstrated neutralization in the GP194-43 peptide hyperimmune sera of immunized rabbits. Notably, IFN-γ production could be effectively promoted in canine macrophages in relation to the activation of macrophages and adaptive immune responses. The results of this study indicate the potential for the use of this immunogen in further investigations involving immunized and infected dogs as E. canis host species.

Antimicrobial drug resistance profile of isolated bacteria in dogs and cats with urologic problems at Chiang Mai University Veterinary Teaching Hospital, Thailand (2012-2016).

The present study aimed to estimate the proportion of bacterial urinary tract infection (UTI) in dogs and cats, assess risks associated with bacterial UTI, and to determine bacterial isolates' antimicrobial susceptibility and resistance pattern from the urinary tract of dogs and cats with urologic problems. The medical records from animals visiting Chiang Mai University Small Animal Veterinary Teaching Hospital between January 2012 and December 2016 were reviewed. In total, 203 dogs and 49 cats with urinary tract diseases that had samples submitted for bacterial culture were identified;198 and 24 bacterial isolates were recovered from dogs' and cats' submitted samples, respectively. At least one episode of bacterial UTI was detected in 75.4% (95% CI: 69.4-81.3) of dogs and in 40.8% (95% CI: 26.6-55.1) of cats with UTI and submitted urine cultures. Of 242 submitted urinary samples from dogs and 60 urinary samples from cats, bacteria were identified in 74.0% (95% CI: 68.4-79.5) and 38.3% (95% CI: 26.0-50.6), respectively. The most common pathogen of bacteria positive cultured from dogs was Staphylococcus spp. (30.3%), followed by Escherichia coli (16.7%), and Proteus spp. (13.6%). For cats, the most common pathogen was Pseudomonas spp. (25.0%), followed by E. coli (20.8%) and Proteus spp. (16.7%). Staphylococcus spp. isolates from dogs and Proteus spp. isolates from cats were highly susceptible to Amoxicillin/clavulanic acid (AMC) at 88% and 75%, respectively. Of all isolated bacteria, 67.1% of the bacteria from dogs and 83.3% from cats were multidrug-resistant (MDR). The proportion of MDR-bacterial urinary tract infections in dogs and cats with urologic problems in this study was high. This observation raises concerns regarding the potential of zoonotic transmission of MDR-bacteria from these companion animals. The results suggested that AMC remains a good empirical drug for treating UTIs in dogs in this region.

Ancylostoma ceylanicum: The Neglected Zoonotic Parasite of Community Dogs in Thailand and Its Genetic Diversity among Asian Countries.

Ancylostoma ceylanicum is a zoonotic helminth that is commonly found in domestic dogs and cats throughout Asia but is largely neglected in many countries. This study aimed to confirm the species of hookworm in dogs and soil environments and investigate the evolutionary analyses of A. ceylanicum among Thai and Asian populations. In a total of 299 dog fecal samples and 212 soil samples from 53 temples, the prevalence rates of hookworm infection by microscopic examination were 26.4% (79/299) and 10.4% (22/212) in dog and soil samples, respectively. A PCR-RFLP targeting the ITS region was then utilized to identify the hookworm species. In dogs, A. ceylanicum was the main hookworm species, and the rates of A. ceylanicum and A. caninum infections were 96.6% and 3.5%, respectively. The genetic characterization and diversity indices of the A. ceylanicumcox1 gene among Thai and Asian populations were evaluated. Nine haplotypes were identified from Thai A. ceylanicum, in which the haplotype diversity and the nucleotide diversity were 0.4436 and 0.0036, respectively. The highest nucleotide diversity of Chinese A. ceylanicum populations suggested that it could be the ancestor of the populations. Pairwise fixation indices indicated that Thai A. ceylanicum was closely related to the Malaysian population, suggesting a gene flow between these populations. The temples with hookworm-positive dogs were associated with the presence of hookworm-contaminated soil, as these levels showed an approximately four-fold increase compared with those in temples with hookworm-negative dogs (OR = 4.38, 95% CI: 1.55-12.37). Interestingly, the genotypes of A. ceylanicum in the contaminating soil and infecting dogs were identical. Therefore, increased awareness and concern from the wider public communities with regard to the responsibility of temples and municipal offices to provide proper deworming programs to community dogs should be strongly endorsed to reduce the risk of the transmission of this zoonotic disease. In addition, parasitic examination and treatment should be strongly implemented before dogs are imported and exported worldwide.

Molecular detection of pathogens in ticks and fleas collected from companion dogs and cats in East and Southeast Asia.

BACKGROUND: Ticks and fleas are considered amongst the most important arthropod vectors of medical and veterinary concern due to their ability to transmit pathogens to a range of animal species including dogs, cats and humans. By sharing a common environment with humans, companion animal-associated parasitic arthropods may potentially transmit zoonotic vector-borne pathogens (VBPs). This study aimed to molecularly detect pathogens from ticks and fleas from companion dogs and cats in East and Southeast Asia. METHODS: A total of 392 ticks and 248 fleas were collected from 401 infested animals (i.e. 271 dogs and 130 cats) from China, Taiwan, Indonesia, Malaysia, Singapore, Thailand, the Philippines and Vietnam, and molecularly screened for the presence of pathogens. Ticks were tested for Rickettsia spp., Anaplasma spp., Ehrlichia spp., Babesia spp. and Hepatozoon spp. while fleas were screened for the presence of Rickettsia spp. and Bartonella spp. RESULT: Of the 392 ticks tested, 37 (9.4%) scored positive for at least one pathogen with Hepatozoon canis being the most prevalent (5.4%), followed by Ehrlichia canis (1.8%), Babesia vogeli (1%), Anaplasma platys (0.8%) and Rickettsia spp. (1%) [including Rickettsia sp. (0.5%), Rickettsia asembonensis (0.3%) and Rickettsia felis (0.3%)]. Out of 248 fleas tested, 106 (42.7%) were harboring at least one pathogen with R. felis being the most common (19.4%), followed by Bartonella spp. (16.5%), Rickettsia asembonensis (10.9%) and "Candidatus Rickettsia senegalensis" (0.4%). Furthermore, 35 Rhipicephalus sanguineus ticks were subjected to phylogenetic analysis, of which 34 ticks belonged to the tropical and only one belonged to the temperate lineage (Rh. sanguineus (sensu stricto)). CONCLUSION: Our data reveals the circulation of different VBPs in ticks and fleas of dogs and cats from Asia, including zoonotic agents, which may represent a potential risk to animal and human health.

Prevalence and risk factors of feline lower urinary tract disease in Chiang Mai, Thailand.

Feline lower urinary tract disease (FLUTD) is a common problem in cats. The objectives of the study were to determine the prevalence, clinical signs, and causes of FLUTD and the risk factors for FLUTD. The medical records of 3486 cats visiting Chiang Mai University Small Animal Veterinary Teaching Hospital (VTH) between November 2016 and October 2017 were reviewed. An age-matched case-control study was performed to determine the risk factors for FLUTD by comparing 78 cats with FLUTD and 78 clinically normal age-matched cats. For each animal, potential risk data were obtained from medical records and cat owner interviews; these were analysed for associations with FLUTD. Multivariable logistic regression analysis was performed to estimate the odds ratios and to adjust for expected confounding factors. The prevalence of FLUTD in cats visiting the Chiang Mai University Veterinary Teaching Hospital was 2.2%. The most common clinical signs identified were urethral obstruction (55.1%) and haematuria (23.1%). The most common diagnoses were feline idiopathic cystitis (FIC) (57.7%) and urolithiasis (struvite) (18%). The multivariable logistic regression analysis results indicated that FLUTD was most likely to be diagnosed in castrated male cats. FIC and urolithiasis were the most common diagnoses in cats with FLUTD, and male sex and castration increased the risk of FLUTD.

Seasonal distributions and other risk factors for Giardia duodenalis and Cryptosporidium spp. infections in dogs and cats in Chiang Mai, Thailand.

The objectives of this study were to explore risk factors associated with Giardia and Cryptosporidium infections in dogs and cats in Chiang Mai, Thailand, to describe the seasonal distributions of Giardia and Cryptosporidium prevalence, and to determine the potential for zoonotic transmission through genetic characterization of isolates. Fecal samples from 301 dogs and 66 cats were collected between August 2009 and February 2010. The presence of Giardia cysts and Cryptosporidium oocysts was determined using zinc sulfate centrifugal flotation and immunofluorescent assay (IFA). Genotype/species were determined by DNA sequence analyses of PCR products from Giardia glutamate dehydrogenase (gdh), beta-giardin (bg), and triosephosphateisomerase (tpi) and Cryptosporidium heat shock protein 70KDa (hsp70) and small subunit-rRNA (SSU-rRNA) genes. Information related to specific risk factors was collected from owners of each animal using a questionnaire. The risk factor data were analyzed for associations with Giardia and Cryptosporidium infections using logistic regression. The overall estimated prevalence of Giardia and Cryptosporidium in dogs was 25.2% and 7.6%, respectively and in cats, 27.3% and 12.1%, respectively. The estimated prevalence of Giardia infection in dogs in the rainy season (31.7%) was significantly higher than in the drier, winter season (17.2%) (p < 0.01). The estimated prevalence of Cryptosporidium infection in dogs and of Giardia and Cryptosporidium infections in cats was not associated with season (p > 0.05). Multivariable analysis indicated that Giardia cysts were more likely to be detected in fecal samples of dogs that resided in high-density environments, drank untreated water, were shedding Cryptosporidium oocysts, were having acute diarrhea or a history of chronic diarrhea, and were collected in the rainy season. All 19 Giardia PCR positive samples typed as G. duodenalis canine adapted genotypes (assemblages C or D). In cats, of six Giardia PCR positive samples, five typed as dog assemblages and one typed as assemblage AI. Of ten dogs with Cryptosporidium PCR positive samples, eight typed as C. canis, one as C. parvum (a zoonotic species) and one had both C. canis and C. parvum. Of three Cryptosporidium PCR positive samples in cats, one typed as C. felis and two typed as C. parvum. The presence of zoonotic G. duodenalis assemblage AI in a cat, and C. parvum in feces of dogs and cats suggests a potential role for a reservoir for zoonotic transmission. Whether or not these presences were from exposure to other animal or human hosts or environment are needed to be confirmed.

Comparative efficacy of afoxolaner and ivermectin in dogs naturally infested with Rhipicephalus sanguineus sensu lato: A clinical field study conducted in Thailand.

Afoxolaner is a novel insecticidal and acaricidal of the isoxazoline family, which is used in veterinary practice to control infestation of dogs by fleas, ticks, and mites (NexGard, Boehringer Ingelheim). Ivermectin is an avermectin administered at microdoses to prevent infection of dogs with Dirofilaria immitis and is used off-label to control Rhipicephalus sanguineus infestation of dogs in numerous countries, including Thailand. Here we conducted two trials to assess the efficacy of afoxolaner for treating natural R. sanguineus sensu lato (s.l.) infestations of dogs residing in the Chiang Mai area of Thailand. The first trial compared the efficacies of afoxolaner and ivermectin in dogs infested with <500 ticks. A randomized, investigator-blinded, controlled study was conducted of 16 dogs, allocated into the groups as follows: afoxolaner (2.7-6.9 mg/kg, PO, group 1; n = 8), ivermectin (300 µg/kg, SC, group 2; n = 5), untreated (group 3; n = 3). Tick counts and drug administration were performed on days 0, 28, and 56. Mean numbers of ticks on day 0 in groups 1, 2, and 3 were not significantly different (225, 169, and 123, respectively; p = .36). The mean number of ticks (%efficacy to control) in groups 1, 2 and 3 on day 28 were 7 (97.05%), 230 (2.95%), and 237, respectively; on day 56 were 4 (96.11%), 93 (9.71%), and 103, respectively; and on day 84 were 1 (98.65%), 44 (40.54%), and 74, respectively. The efficacy of afoxolaner was >96%, whereas the efficacy of ivermectin was significantly lower compared with that of afoxolaner (p < .05) and never achieved the 90% efficacy threshold claimed by registration agencies. The second trial assessed the efficacy of afoxolaner for treating dogs with heavy tick infestations (>500 ticks/dog), including four dogs from two households. The dogs were treated monthly with Afoxolaner. The mean values of the numbers of ticks on dogs in the 2 households were not significantly different (913 and 800 on day 0, p = .18). The numbers of ticks significantly decreased thereafter, and the efficacy of afoxolaner was >99% on days 28, 56, and 84. Adverse reactions were not observed in either trial. In conclusion, this study confirms the efficacy of afoxolaner against adult R. sanguineus s.l. that naturally infests dogs that inhabit Thailand.

Bactericidal Effect of Clove Oil against Multidrug-Resistant Streptococcus suis Isolated from Human Patients and Slaughtered Pigs.

Streptococcus suis is a zoonotic pathogen that is currently considered an emerging multidrug-resistant (MDR). Increasing antibiotic resistance can lead to the unsuccessful treatment of S. suis infection. Recently, many investigations of medicinal plants were conducted for the treatment of infection as a result of the increase of antibiotic-resistant bacteria. The aims of this study were to determine the chemical composition of essential oil from Syzygium aromaticum (L.) Merr. & L.M. Perry and the antibacterial activities of clove oil on MDR S. suis. Using gas chromatography coupled to a mass spectrometer, eugenol (97.76%) was found to be the major active ingredient of clove oil. In vitro antibacterial activities of clove oil against MDR S. suis were evaluated. Using the agar disc diffusion test, the clove oil showed a maximum zone of inhibition at 15% (v/v) oil concentration. In a broth microdilution method, the minimum bactericidal concentration of clove oil against all MDR S. suis isolates was 0.1% (v/v). A time-kill analysis was performed, and the killing kinetics of clove oil showed that MDR S. suis was completely reduced after 15 min of exposure to clove oil. In addition, clove oil exhibited a strong antibacterial activity at all pH values applied following incubation of MDR S. suis in pH-adjusted media with clove oil. Moreover, scanning electron microscopy revealed the nonviable S. suis isolates clearly showed atypical form and cell membrane lysis after incubation with clove oil. This study confirms the efficacy of clove oil as a natural antimicrobial against MDR S. suis and suggests the possibility of employing it as a promising alternative product for control of infectious diseases caused by S. suis in animal and human patients.

Intestinal Parasites and the Occurrence of Zoonotic Giardia duodenalis Genotype in Captive Gibbons at Krabokkoo Wildlife Breeding Center, Thailand.

Intestinal parasitic infections can have an impact on health and growth of wildlife. The current study aims were to determine the prevalence of intestinal parasites and to molecular characterize Giardia duodenalis and Cryptosporidium spp. in captive gibbons at Krabokkoo Wildlife Breeding Center, Thailand. Fifty-five gibbons, 2 agile- (Hylobates agilis), 38 lar- (Hylobates lar) and 15 pileated gibbons (Hylobates pileatus) were included in this study. Fecal samples were collected individually at Krabokkoo Wildlife Breeding Center, Chachoengsao province, eastern Thailand, in November 2013. Intestinal parasitic infections were examined by zinc sulfate centrifugation flotation and by a commercially available immunofluorescent assay (IFA) for detection of G. duodenalis and Cryptosporidium spp.. Polymerase chain reaction targeting the Giardia glutamate dehydrogenase (gdh), beta- giardin (bg), triose phosphate isomerase (tpi) genes, and the Cryptosporidium small subunit-rRNA and heat-shock protein (hsp70) following by DNA sequencing were performed on the IFA positive samples. The overall prevalence of intestinal parasitic infection in gibbons at Krabokkoo Wildlife Breeding Center was 12.7% (95%CI: 5.3-24.5), Strongyloides spp. eggs or larvae were present in all positive samples. Co-infections with G. duodenalis were detected in 1.8% (95%CI: 0.1-9.7) of the samples. Based on the sequencing results of the three genes, the IFA Giardia positive isolate typed as the zoonotic genotype B. Since the data reveals the occurrence of zoonotic Giardia genotype, good hygiene management is suggested to prevent the transmission of this pathogen from gibbon to human, and vice versa.

The Effectiveness of a Foot and Mouth Disease Outbreak Control Programme in Thailand 2008⁻2015: Case Studies and Lessons Learned.

Three Foot and Mouth Disease (FMD) outbreaks in northern Thailand that occurred during the implementation of the national FMD strategic plan in 2008⁻2015 are described to illustrate the lessons learned and to improve the prevention and control of future outbreaks. In 2008, during a FMD outbreak on a dairy farm, milk delivery was banned for 30 days. This was a part of movement management, a key strategy for FMD control in dairy farms in the area. In 2009, more than half the animals on a pig farm were affected by FMD. Animal quarantine and restricted animal movement played a key role in preventing the spread of FMD. In 2010, FMD infection was reported in a captive elephant. The suspected source of virus was a FMD-infected cow on the same premises. The infected elephant was moved to an elephant hospital that was located in a different province before the diagnosis was confirmed. FMD education was given to elephant veterinarians to promote FMD prevention and control strategies in this unique species. These three cases illustrate how differences in outbreak circumstances and species require the implementation of a variety of different FMD control and prevention measures. Control measures and responses should be customized in different outbreak situations.

Two different genogroups of Ehrlichia canis from dogs in Thailand using immunodominant protein genes.

Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis (CME). While there is a high prevalence of CME in Thailand, genetic diversity of E. canis is still poorly defined. This study examined the molecular characteristics of E. canis using PCR and phylogenetic analysis of the dsb, gp19 and gp36 genes. DNA was extracted from 220 whole blood samples of naturally infected dogs, and all had clinical signs compatible with tick-borne diseases. Of these, 16.4% (36/220) provided positive E. canis DNA via the dsb and gp19 genes. However, only 13 out of the 36 samples (36.1%) were positive for the gp36 gene. Sequences of the dsb gene had very high identity (99-100%) with previously deposited E. canis sequences. Sequences of the gp19 gene were similar to those from US and Taiwanese genogroups (98.8-99.5% identity). Elucidation of genetic characteristics of E. canis based on the gp36 gene displayed 91.4-99.1% shared identity. There were 426-429 bp of a 5' end pre-repeat tandem region, a 27 bp repetition with variable numbers of a tandem repeat (TR) region of 9 amino acid sequences (TEDSVSAPA), and a variable 3' end region with sequence length depending on the isolate (72-93 bp). Phylogenetic trees of E. canis, particularly using the gp36 amino acid sequences, showed that the Thai strains fell into two phylogenetic clades contained within other worldwide E. canis strains. Alignment and phylogenetic analysis suggested that E. canis strains from Thailand could be divided into two genogroups, the US and Taiwanese genogroups. This study provides the first characterization of the dsb and gp19 genes of E. canis in Thailand, the results support the conclusion that the gp36 is a potential target for genotyping and elucidation of phylogenetic relationships among E. canis strains.

Prevalence and Multilocus Genotyping Analysis of Cryptosporidium and Giardia Isolates from Dogs in Chiang Mai, Thailand.

The occurrence and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis isolated from dogs in Chiang Mai, Thailand were determined. Fecal samples were collected from 109 dogs between July and August 2008. Cryptosporidium spp. infection was determined by immunofluorescent assay (IFA), PCR assays that amplify Cryptosporidium heat-shock protein 70 kDa (hsp70), and two PCR assays that amplify a small subunit-ribosomal RNA (SSU-rRNA). Giardiaduodenalis infection was identified using zinc sulfate centrifugal flotation, IFA, and four PCR assays that amplify the Giardia glutamate dehydrogenase (gdh), beta-giardin (bg), and generic and dog-specific assays of triosephosphate isomerase (tpi) genes. Overall prevalence of Cryptosporidium spp. and G.duodenalis was 31.2% and 45.9%, respectively. Sequence analysis of 22 Cryptosporidium-positive samples and 21 Giardia-positive samples revealed the presence of C.canis in 15, and C. parvum in 7, G. duodenalis Assemblage C in 8, D in 11, and mixed of C and D in 2 dogs. Dogs in Chiang Mai were commonly exposed to Cryptosporidium spp. and G. duodenalis. Cryptosporidium parvum can be isolated from the feces of dogs, and all G. duodenalis assemblages were dog-specific. Dogs could be a reservoir for a zoonotic Cryptosporidium infection in humans, but further studies will be required to determine the clinical and zoonotic importance.

Molecular characterization of canine parvovirus in Vientiane, Laos.

The global emergence of canine parvovirus type 2c (CPV-2c) has been well documented. In the present study, 139 rectal swab samples collected from diarrheic dogs living in Vientiane, Laos, in 2016 were tested for the presence of the canine parvovirus (CPV) VP2 gene by PCR. The results showed that 82.73% (115/139) of dogs were CPV positive by PCR. The partial VP2 gene was sequenced in 94 of the positive samples; 91 samples belonged to CPV-2c (426Glu) subtype, while 3 samples belonged to the CPV-2a (426Asn) subtype. Notably, phylogenetic analysis of amino acid sequences revealed a close relationship between Laotian isolates and novel Chinese CPV-2c isolates. In Laotian CPV isolates, aligned protein sequences indicated a high rate of residue substitutions at positions 305, 324, 345, 370, 375, and 426 in the GH loop. The mutation at residue 370 (Q370R), a single mutation, was characterized as a unique mutant residue specific to the Laotian CPV-2c variant.

Comparisons of mammalian Giardia duodenalis assemblages based on the ß-giardin, glutamate dehydrogenase and triose phosphate isomerase genes.

The objective of this study was to determine and compare the assemblages of Giardia duodenalis isolated from mammalian fecal samples using the ß-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes. A total of 202 samples, either submitted to the Veterinary Diagnostic Laboratory (Parasitology) at Colorado State University or part of ongoing research studies, were typed. A subset of 50 dog samples were also assessed by the tpi-D-specific primers. Of these, 183 were from dogs, 13 were from cats, two were from llamas, and one each was from a calf, an alpaca, a sheep, and a horse. The majority of the dogs (171 of 183 isolates) in this study were infected with only dog-adapted Assemblage C or D. The tpi-D-specific primers confirmed that 28 of the samples that typed as Assemblage D by the bg and gdh genes were also Assemblage D by the tpi-D-specific primers. Only 12 isolates were Assemblage A alone or Assemblage A and Assemblage C or D. Of the 13 cat isolates, seven were Assemblage F, two were Assemblage D, three were Assemblage A and 1 contained both Assemblages C and D. The calf isolate was Assemblage E (gdh, tpi) and the alpaca (bg, gdh), llamas (gdh), sheep (bg, gdh, tpi) and horse (tpi) isolates were all Assemblage A. When the assemblage could be determined for more than one gene, 91 of 117 dog isolates gave consistent results and 8 of 9 cat isolates gave consistent results.