A parasitological survey and the molecular detection of Entamoeba species in pigs of East Java, Indonesia.

Background and Aim: In several countries, two Entamoeba porcine species, Entamoeba suis and Entamoeba polecki (subtype 1 and 3), have been detected using molecular methods and identified pathogenicity associated with enteritis. However, globally, Entamoeba infection prevalence in pigs is extremely limited. This study aimed to coprologically and genetically examine pig parasites to estimate prevalence of Entamoeba in three pig farms in East Java, Indonesia. Materials and Methods: Hundred porcine fecal samples (Landrace) were collected from three East Javan farms in well-known swine industry regions. Fecal samples were examined under a microscope after sugar-flotation centrifugation, and molecular species and subtype identification were performed using polymerase chain reaction (PCR) and primer pairs targeting small-subunit ribosomal RNA. Results: Microscopy examinations identified parasites in 89/100 fecal samples; Entamoeba spp. cysts were the most frequent in these samples. Polymerase chain reaction showed that 58 samples were comprised of mixed Entamoeba suis and Entamoeba polecki, 22 E. suis alone, and nine E. polecki alone infections. Epolec F6-Epolec R6 primers successfully amplified E. polecki ST1-4 subtypes, while Epolecki 1-Epolecki 2 amplified only the E. polecki ST1 subtype. Entamoeba polecki ST1-specific primers successfully detected the ST1 subtype in 19/67 E. polecki positive samples. Conclusion: Entamoeba spp. prevalence in Indonesian pigs was previously shown to be high. On coprological examination of East Javan pigs, we detected high Entamoeba spp. levels, in which we genetically identified as E. suis (80.0%), E. polecki (67.0%), and E. polecki ST1 (19%).

Synthesis and cytotoxic activities of 8- and 6-demethyleucalyptins.

Secondary metabolites in plants influence the health of herbivores such as Japanese rock ptarmigans that feed on the leaves and fruits of alpine plants. Thus, it is important to understand the secondary metabolites of alpine plants and their biological activities for conserving Japanese rock ptarmigans. We isolated C-methylflavone from the leaves of Kalmia procumbens, on which Japanese rock ptarmigans feed. Although its structure was deduced to be 8-demethyleucalyptin by comparing its nuclear magnetic resonance (NMR) data with the reported ones, the possibility that the isolated compound is 6-demethyleucalyptin cannot be ruled out. Thus, both isomers were synthesized. The isolated compound was unambiguously determined to be 8-demethyleucalyptin by comparing its NMR data with those of the synthetic ones. Cytotoxic evaluation of 8- and 6-demethyleucalyptins revealed that only the former showed cytotoxicity against HCT116 and MRC-5 cells. The present study provides not only easy access to 8- and 6-demethyleucalyptins, but also their biological information.

Reduction of macrophages by carrageenan decreases oocyst output and modifies local immune reaction in chick cecum with Eimeria tenella.

This study aimed to evaluate the disease severity and local immune responses in macrophage-depleted chicks with Eimeria tenella. Macrophages were reduced by intraperitoneal injection of a carrageenan solution at 12, 13, and 16 days old, whereas the control group received intraperitoneal phosphate-buffered saline. Both chick groups were orally inoculated with E. tenella sporulated oocysts at 14 days old. Feces were collected daily, which were then quantified for oocysts. The chicks were sacrificed on day 5, and the ceca were collected for histopathological observation. The gene expression levels were measured using real-time quantitative reverse transcription-polymerase chain reaction. Macrophage-depleted chicks have been observed to shed a significantly reduced number of fecal oocysts compared to the infected control group. The parasite burden score in cecum specimens of macrophage-depleted chicks was significantly lower than those of infected control on day 5 after infection. Furthermore, macrophage reduction yielded significantly lower cecum histopathological scores and CD4 expression than those of the infected control group. The expression of interleukin (IL)-18, IL-22, interferon-γ, and inducible nitric oxide synthase was also noted to be significantly upregulated in both infected control and macrophage-depleted chicks compared to uninfected chicks. IL-4, IL-13, IL-17, and perforin expressions were also higher with macrophage depletion than in both control groups. These results suggest that macrophages serve as an invasive gate or a transporting vehicle to the site of first merogony. Furthermore, mononuclear phagocytes may play an important role in local immune responses, thus contributing to parasite development during early E. tenella infection.

Occurrence and genetic identifications of porcine Entamoeba, E. suis and E. polecki, at Tangerang in West Java, Indonesia.

Entamoeba suis and E. polecki subtype (ST) 1 and ST3 recently have been inferred to be virulent in pigs. However, because relevant molecular epidemiological surveys have been limited, the prevalences of these species remain unknown and their pathogenicities are still controversial. We surveyed 196 fecal samples of pigs (118 of adults, 78 of piglets) at Tangerang in West Java, Indonesia, in 2017, employing PCR using porcine Entamoeba-specific primers. E. suis was the more frequently detected species, observed in 81.1% of samples, while E. polecki ST1 and ST3 were detected in 18.4% and 17.3% of samples, respectively; mixed infections (harboring 2-3 species or subtypes of Entamoeba) were confirmed in 29.3% of positive samples. Statistically significant differences in the positive rates were not seen between adult pigs and piglets, except for those of E. polecki ST3. The prevalences of Eimeria spp. and/or Cystoisospora suis (79.1%), strongyles (55.6%), and Strongyloides spp. (6.1%) were also observed morphologically in the samples. Further chronological or seasonal investigations of pigs and humans in these high-prevalence areas are needed to assess the virulence of the Entamoeba parasites, including the effects on pig productivity, and to evaluate the zoonotic impacts of these organisms.

Prevalence of microorganisms associated with feline gingivostomatitis.

OBJECTIVES: Feline gingivostomatitis (FGS) is a painful chronic inflammatory disease of the oral cavity. The purpose of this study was to examine the frequency of detection of certain common feline bacteria and viruses to determine any potential associations with FGS. METHODS: A multicentre case-control study design was conducted. In total, 72 control cats and 32 cats with FGS were included in the study. Oral swabs were cultured for bacterial identification and a PCR assay was carried out to examine the infection of feline calicivirus (FCV), feline herpesvirus-1 (FHV-1), Chlamydia felis, Mycoplasma felis and Bordetella bronchiseptica. RESULTS: There was a significant difference in age distribution between the control and the FGS group. Based on a PCR assay, the positive rate of FCV was significantly higher in FGS cats than control animals. For other infectious pathogens, including FHV-1, C felis and M felis, there was no significant difference. Bacterial culture of oral swabs revealed that Pasteurella multocida was most frequently detected, but the detection rate was significantly lower in FGS cats. In FGS cats, the incidence of Enterococcus faecalis and anaerobic bacteria were more frequently isolated than in control cats. CONCLUSIONS AND RELEVANCE: This study indicates that the positive rate of FCV was significantly higher in cats with FGS, and the microflora of the oral cavity of cats with FGS might be disrupted, although additional studies are required to compare the oral microbiome in cats of a variety of ages.

First detection and molecular identification of Entamoeba bovis from Japanese cattle.

Thus far, Entamoeba species have been classified based on morphology such as the number of nuclei in mature cysts and their hosts. Using recently developed molecular tools, ruminant Entamoeba spp. are currently classified into four species/genotypes: E. bovis and Entamoeba ribosomal lineages (RL) 1, 2, and 4. However, the distribution or pathogenicity of ruminant Entamoeba has not been well documented. In the present study, we examined a total of 25 fecal and seven environmental samples collected from six farms in Japan from 2016 to 2017 by the floatation method and PCR and sequencing analyses. Consequently, we detected Entamoeba cysts in 18 of 25 cattle samples and four of the seven environmental samples, including soil and drinking water, by microscopic examinations. In sequential examinations, Entamoeba-positive cattle were found to shed cysts without any clinical symptoms for more than 8 months. By PCR for molecular identification, isolates in ten cattle and one soil sample were successfully sequenced and formed a cluster of E. bovis, which was separated from those of other Entamoeba species/genotypes such as RL1-4 in phylogenetic analysis. To our knowledge, this is the first report about E. bovis in Japan, and our results may implicate that E. bovis is not pathogenic.

SURVEILLANCE FOR ANTIBODIES AGAINST SIX CANINE VIRUSES IN WILD RACCOONS (PROCYON LOTOR) IN JAPAN.

Raccoons (Procyon lotor) are found worldwide. They are frequently seen in crowded inner cities as well as in forests or wooded areas, often living in proximity to humans and their pets. We examined sera from 100 wild raccoons in Japan for antibodies to six canine viruses with veterinary significance to assess their potential as reservoirs. We also aimed to understand the distribution of potentially infected wildlife. We found that 7% of samples were seropositive for canine distemper virus (CDV), 10% for canine parvovirus type 2, 2% for canine adenovirus type 1, 6% for canine adenovirus type 2, and 7% for canine coronavirus. No samples were found to be seropositive for canine parainfluenza virus. Seropositivity rates for canine distemper virus and canine parvovirus type 2 were significantly different between areas, and younger raccoons (<1 yr old) were more frequently seropositive than older raccoons. Because raccoons belong to the suborder Caniformia, similar to dogs (Canis lupus familiaris), our results suggest that they can act as reservoirs for some of these important canine viruses and might be involved in viral transmission. Further study should include isolation and analysis of canine viruses in wild raccoons from a wider area.

Development of molecular diagnostic protocols for detecting three types of Entamoeba from diarrheal and asymptomatic pigs and environmental moist soils.

Entamoeba suis and Entamoeba polecki subtypes (ST) 1 and 3 have recently been implicated in disease outbreaks in pigs. However, the distributions of these parasites in Japan and the potential sources of infection on farms still remain unclear. Here, we examined a farm of fattening/growing pigs with abnormal feces in Kagoshima Prefecture, Japan, and found the presence of parasites in the farm environment. Examination of intestinal tissues from pigs presenting with ulcerative colitis revealed a large number of trophozoites that had invaded the lesions. We identified single and mixed infections of E. suis and E. polecki ST1 and ST3 in paraffin sections or fecal samples from affected pigs. Two subtypes of Entamoeba were identified using four primer sets by PCR and sequencing. The parasites were detected in moist soil samples obtained around the drinking water source or puddles, implicating transmission of cysts via contaminated soils. Additionally, we found evidence of Entamoeba spp. and coinfections in surveyed pigs without any diarrhea at two neighboring farms. Our results establish methods for successfully identification of parasites, including cases in which multiple infections are present.

Report of fatal mixed infection with Cryptosporidium parvum and Giardia intestinalis in neonatal calves.

In the production and management of beef and dairy cattle, controlling diarrhea is one of the important concerns. Pathogenic agents of the disease, protozoan parasites including Cryptosporidium spp., are difficult to control, making prevention, diagnoses, and treatment of diarrhea. In the present study, we investigated a farm with a history of calf deaths over a period of 10 years in order to determine the cause of disease and to clarify the detailed distribution of the pathogens. In four examined calves that were reared in calf pens, all were positive with Cryptosporidium and/or Giardia, while the other breeding stock and adult cattle were negative. Molecular analyses revealed that the isolates from calves were C. parvum subtype IIaA15G2R1 as a zoonotic and G. intestinalis assemblage E. Other pathogenic bacteria and diarrhea-causing viruses were not detected. After treating the calf pens with boiling water and milk of lime (Ca[OH]2), oocysts of C. parvum and cysts of G. intestinalis were not found and no additional calves died. This is the first report to describe the mixed infection of both parasites in Japan.

First report of molecular identification of Cystoisospora suis in piglets with lethal diarrhea in Japan.

Cystoisospora suis is a pathogen that causes diarrhea in pigs and can lead to serious disease. Species identification, especially by histopathological examination, is often difficult because of morphologically similar parasites such as Eimeria species. In this study, we used histopathological, bacteriological, virological, and parasitological methods to identify the cause of the disease in two piglets with severe diarrhea. Villous atrophy, diffuse necrosis, and flattening of mucosal epithelial cells were found in the ilea of examined piglets, and coccidian parasites were found in the cytoplasm of the epithelial cells. In some merozoites in the meronts, the presence of two nuclei indicated type 1 merozoites, characteristic of C. suis. According to Cystoisospora-specific PCR targeting the rRNA internal transcribed spacer 1 (ITS1) gene, the sequences of the products were 98.5% similar to those of C. suis. Escherichia coli (O149 serogroup) exhibiting a virulence factor profile (LT, STb, and EAST1 as toxins and F4 as a colonization factor) was detected in one piglet. No other bacteria or significant enteric viruses were found. Co-infection with C. suis and E. coli could imply aggravation of the disease, although further study is needed to assess the pathogenicity of this interaction. This study is the first to clarify by molecular analysis the sequences of C. suis detected in piglets in Japan.

Localization of heat shock protein 110 in canine mammary gland tumors.

Heat shock proteins (HSPs) function as molecular chaperones in the regulation of protein folding, conformation, and assembly; in addition, they also protect cells from protein-protein aggregation resulting from cellular stress. Recently, HSPs were shown to be overexpressed in several human cancer cells compared with normal cells. HSPs are considered to be related to apoptosis-associated proteins, and inhibition of apoptosis promotes tumor growth. Canine mammary gland tumors have received a great deal of attention from researchers due to the many common biological and histological characteristics that they share with human tumors. We previously confirmed that HSP110 is a canine mammary gland tumor antigen and reported that HSP110 mRNA expression significantly increased in tumor tissue. We have now created a functional recombinant canine HSP110 protein and a rabbit anti-HSP110 polyclonal antibody. This recombinant protein can refold heat-denatured firefly luciferase at 42°C. Immunohistochemical analysis showed that HSP110 was mainly localized in the cytoplasm of epithelial and interstitial cells in canine mammary gland tumors. Extensive genomic research has revealed genetic similarities between humans and dogs; comparative oncological studies between these species have made remarkable progress. The results reported here contribute valuable oncological knowledge for the development of novel therapeutic methods in both veterinary science and human medicine.

Ultrastructural characteristics and molecular identification of Entamoeba suis isolated from pigs with hemorrhagic colitis: implications for pathogenicity.

Protozoan parasites of the genus Entamoeba infect many classes of vertebrates and are primarily classified based on morphological criteria. To date, only a few species have been proven to cause disease. Here, we examined the pathology of infected pigs with hemorrhage and detected Entamoeba parasites. Isolates were characterized genetically and ultrastructurally to identify the species. Histopathologically, bleeding and thrombus formation were seen only in the large intestine mucosa, where a large number of trophozoites or some Entamoeba cysts were observed around breakdowns in the lamina propria. No screw-shaped bacteria were detected in the lesions, and no pathogenic bacteria such as Brachyspira spp. were detected in fecal cultures. Interestingly, electron microscopy revealed that the parasites possessed mitochondrial organelles, unlike other Entamoeba spp. The isolates were identified as Entamoeba suis by PCR analysis and sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. In phylogenetic analyses based on the actin gene, the E. suis isolate formed a cluster with Entamoeba histolytica and Entamoeba invadens, as well as with other parasites of the Amoebidae. Whether the pathogenicity of the E. suis isolate is affected by the severity of infection or host health status remains unclear; however, our results suggest that E. suis could cause or exacerbate clinical symptoms such as hemorrhagic colitis or diarrhea.

Host specificity and in vivo infectivities of the mouse coccidian parasites Eimeria krijgsmanni.

In the present study, infection experiments of E. krijgsmanni using various hosts were conducted to elucidate the host specificity among some animals and the infectivity to mouse strains. According to the results, the infection was not found in most animals, except for rats, in which some oocyst shedding was detected, and there was no significant difference in infectivity among mouse strains. Additionally, oocyst shedding was hardly detectable in a secondary infection to immunocompetent mice, although it was found in immunodeficient mice. These results indicated that only immunocompetent mice could develop adaptive immunity against reinfection by stimuli of the primary infection. Furthermore, the infection experiments were performed with splenic macrophage (Mφ)-depleted mice with a reagent and Beige (Bg) mice known to be a strain of mice with low NK cell activity. No significant effect was found in primary or secondary infections in the Mφ-depleted mice, whereas the mortality rate was clearly increased in Bg mice inoculated with a large number of oocysts. Their oocyst shedding was similar to that of immunocompetent hosts. Taken together, these results suggested that Mφ has only a minor role in the immune response, but the NK cell has an important function in resistance to primary infection of E. krijgsmanni.

Localization of eimeripain, an Eimeria tenella cathepsin B-like cysteine protease, during asexual and sexual intracellular development in chicken ceca.

Hemorrhagic diarrhea in poultry is caused by Eimeria tenella, the most pathogenic avian coccidian parasite, and new approaches to treat the disease are continually being sought. Although eimeripain, a cathepsin B-like cysteine protease from E. tenella, has recently been identified as a novel anticoccidial drug target, its localization during the intracellular development of parasites remains unclear. Here, we demonstrate the expression of eimeripain during asexual and sexual development of E. tenella in vivo. Promature eimeripain was detected only in the early immature second generation of schizonts. In contrast, the mature eimeripain was most strongly detected in the middle-sized immature second generation of schizonts. Both promature and mature eimeripain disappeared depending on the maturation level of second generation of schizonts, but were strongly expressed again in the third generation of schizonts. In the sexual stage, both promature and mature eimeripain were detected in the cytoplasm of micro- and macro-gametocytes and zygotes, but expression became weak in zoites forming oocysts. Collectively, our findings suggest that eimeripain might play a key role in the differentiation of intracellular zoites in the ceca and could be an interesting candidate to develop a novel, effective anti-coccidian drug.

Elongation factor-1α is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum.

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-ß- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.

HlCPL-A, a cathepsin L-like cysteine protease from the ixodid tick Haemaphysalis longicornis, modulated midgut proteolytic enzymes and their inhibitors during blood meal digestion.

Ticks employ a battery of proteases to digest the contents of host blood meals. Host hemoglobin degradation is facilitated by proteolytic networks in the midgut, the first major region of the body where ingested blood comes into contact with the tick's internal tissues. Our previous studies indicated that HlCPL-A, a cathepsin L-like cysteine protease isolated from the midgut of the ixodid tick Haemaphysalis longicornis, is a potent hemoglobinase, and plays important roles in the digestion of blood acquired from a host. In this paper, we report the effects of silencing of the HlCPL-A gene in H. longicornis using RNA interference (RNAi). We observed that the survival of HlCPL-A-silenced ticks was reduced compared with that of controls during blood digestion, most likely due to the compromised ability of ticks to digest blood. The morphological analysis results of midgut lumen were different between HlCPL-A-silenced ticks and controls, indicating that HlCPL-A plays a crucial role in hemolysis in the midgut of ticks. The expression level was analyzed using quantitative RT-PCR-based endogenous expression approach. Compared to that in malE double stranded RNA (dsRNA)-treated ticks, in the midgut of HlCPL-A dsRNA-treated ticks, some proteases and inhibitors related to the hemoglobin digestive cascade were up-regulated while the others were down-regulated. These results suggest that HlCPL-A is related to the multi-enzyme cascade and protease network for hemoglobin digestion. These findings suggest that the hemoglobin digestive cascade may assemble in the midgut of ticks.

A hemocyte-derived Kunitz-BPTI-type chymotrypsin inhibitor, HlChI, from the ixodid tick Haemaphysalis longicornis, plays regulatory functions in tick blood-feeding processes.

Inhibitors of proteases play key roles in the biological processes of vertebrate and invertebrate animals, including arthropod parasites. Here, we describe a cDNA that encodes a functionally active chymotrypsin inhibitor of the BPTI/Kunitz family of serine protease inhibitors from the hemocytes of the ixodid tick, Haemaphysalis longicornis, herein called HlChI. HlChI sequence is evolutionarily conserved and contains six cysteine residues and three disulfide bonds with a calculated molecular weight of 9.1 kDa. HlChI-specific mRNA was expressed in all developmental stages of ticks and the expression was up-regulated by host's blood-feeding processes. Endogenous HlChI was localized mainly in the hemocytes. HlChI potently inhibited bovine pancreatic α-chymotrypsin for hydrolyzing the fluorogenic substrate (IC(50) 8.32 nM, K(d) 5.35 ± 1.01 nM) and bovine casein digestion. However, HlChI weakly inhibited bovine pancreatic trypsin and could not affect the porcine elastase activity, suggesting its narrow specificity to chymotrypsin. HlChI was stable over the pH range 2-11 and heating up to 70 °C at pH 8. HlChI was highly stable to 8 M urea and 2% SDS at pH 8.0, when treated for 24 h at 37 °C. However, 0.2 M 2-mercaptoethanol caused complete but reversible inactivation of HlChI. Knockdown of HlChI gene by RNA interference (RNAi) caused death of the feeding ticks, failure of ticks to engorge and significantly reduced body weight gain. RNAi also resulted in significantly decreased egg conversion ratio and fecundity. These results suggest that HlChI is a chymotrypsin-specific inhibitor with high stability and may play regulatory functions in host's blood-feeding processes and tick reproduction.

Phylogenetic identification of Cystoisospora spp. from dogs, cats, and raccoon dogs in Japan.

Cystoisospora spp. from feces in dogs, cats, and raccoon dogs were isolated, sequenced at the small subunit ribosomal RNA gene locus and compared to other Cystoisospora spp. Cystoisospora oocysts from dogs and raccoon dogs were morphologically similar with those of C. ohioensis, and cat isolates were similar with those of C. felis. The sequences from dogs and raccoon dogs, and cats have a homology with C. ohioensis and C. felis, respectively. Phylogenetic analysis of the DNA sequences showed that the dog and raccoon dog isolates were nested in a clade with other Cystoisospora spp. including C. ohioensis, C. belli, and C. orlovi. The cat isolate formed a sister group with C. felis that was a separate clade from the dog and raccoon dog group. We report sequence variation in these Cystoisospora sequences and have identified raccoon dogs as another carnivore host for Cystoisospora spp. infecting dogs.

Effect of low pH on the morphology and viability of Cryptosporidium andersoni sporozoites and histopathology in the stomachs of infected mice.

The genus Cryptosporidium includes many common parasites infecting animals and humans, and is a major cause of diarrheal illness worldwide. The biology of gastric Cryptosporidium spp., including replication in the stomach, has not been well documented. This study evaluated the viability of Cryptosporidium andersoni sporozoites in gastric environments after excystation and examined the endogenous development and histopathological changes in the stomachs of infected mice, using a novel type of C. andersoni. Sporozoites were affected by low pH (61.6% viability after 3h at pH2.0). Electron microscopy revealed developmental parasites on the gastric foveolae but not on the surface of the gastric mucosa. Histopathological examinations at 1, 2, 4 and 12 weeks p.i. uncovered three different lesions. The gastric mucosa of foveolae filled with parasites was extended and the amount of neutral mucopolysaccharide at the mucosal surface was decreased with the first type of lesion. The gastric mucosa was atrophied, some gastric glands were disrupted and the amount of acid mucopolysaccharide at the mucosal surface was increased with the second type. Finally, the gastric mucosa was slightly extended and goblet cells were present in the gastric mucosa, indicating intestinal metaplasia, in the third type. No parasites were detected in these areas with increased acidic mucin and indications of metaplasia. The results suggest that C. andersoni parasites could not survive in acidic environments for a long period before invading host cells and preferentially develop in neutral sites of the gastric mucosa, resulting in histopathological changes and chronic shedding of oocysts.

Survey and molecular characterization of Cryptosporidium and Giardia spp. in owned companion animal, dogs and cats, in Japan.

Compared with other countries, surveys of these parasites have been rarely performed in companion animals of Japan in spite of their significance for public health. Here, we investigated pet dogs and cats in Japan for the first time, and genetically analyzed the isolates to evaluate the risk of zoonotic infections. Seventy-seven fecal samples were collected from privately owned dogs and 55 samples from owned cats in Osaka city, Japan. Cryptosporidium oocysts were identified in 3/77 dogs (3.9%) and 7/55 cats (12.7%), and Giardia infection in 2/77 dogs (2.6%) and 1/55 cats (1.8%). Amplification of the target regions for genotyping was successful, Cryptosporidium isolates in dogs and cats were identified as C. canis and C. felis, respectively, and those of Giardia in dogs and cats were G. intestinalis Assemblages D and F. The discharge period of the oocysts varied within 3-16 weeks and that of the cysts was 12 weeks. To date, zoonotic types of both parasites have been identified in other animals in Japan, and further large-scale studies are needed to determine the distribution of zoonotic genotypes in these animals, especially those closely associated with humans.