Specific increase of Fusobacterium in the faecal microbiota of neonatal calves infected with Cryptosporidium parvum.

The faecal microbiota plays a critical role in host health, with alterations in the human faecal microbial composition associated with various conditions, particularly diarrhoeal diseases. However, little is known about microbial changes during cryptosporidiosis, one of the most important diarrhoeal diseases caused by protozoa in cattle. In this study, alterations in the faecal microbiota of neonatal calves as a result of Cryptosporidium parvum infection were investigated on a C. parvum-positive farm. Comparisons were made among groups of C. parvum-infected, rotavirus-infected, and the pathogen-negative calves. A specific increase in the abundance of Fusobacterium was observed in the faecal microbiota of C. parvum-infected animals. Diarrhoea severity increased in accordance with the abundance of C. parvum and Fusobacterium. Moreover, the specific increase of Fusobacterium appeared to be a universal feature of C. parvum infection, since neonatal calves from geographically separated areas showed the same result. These observations indicated that the growth of Fusobacterium may be an important aggravating factor of cryptosporidiosis.

First detection of Allobilharzia visceralis (Schistosomatidae, Trematoda) from Cygnus cygnus in Japan.

Adult schistosomes were detected in the veins or capillaries of the large intestine, mesentery, liver, and adrenal glands in eight of 13 whooper swans (Cygnus cygnus) examined in Iwate Prefecture, Japan. However, neither eggs nor severe tissue injuries were observed in any of the swans. The schistosomes were definitively identified as Allobilharzia visceralis based on the nucleotide sequences of the ribosomal internal transcribed spacer (ITS) region. Allobilharzia visceralis infections have been reported in whooper swan in Iceland and tundra swan (Cygnus columbianus) in North America. These detections suggest that A. visceralis is distributed extensively along the swan flyways because the swans are migratory birds. To the best of our knowledge, this is the first report of A. visceralis infection in Asia.

Molecular epidemiological analyses of Cryptosporidium parvum virus 1 (CSpV1), a symbiotic virus of Cryptosporidium parvum, in Japan.

We show that Cryptosporidium parvum virus 1 (CSpV1), a member of the family Partitiviridae, genus Cryspovirus that can infect Cryptosporidium parvum, is a new candidate for high-resolution tool for tracing C. parvum. CSpV1 was detected in all C. parvum-positive samples tested. Phylogenetic analysis of dsRNA1 sequence from CSpV1 can distinguish infected areas of C. parvum on the national level. Sequences detected in samples from Iwate prefecture and other islands (Tanegashima, and Okinawa) belonged to a single clade. This system can differentiate the samples from Hokkaido and south part of Japan as well as from other countries. Samples from Iwate, Tanegashima, and Okinawa belonged to a single subclade, respectively. Therefore, the CSpV1 dsRNA sequences reflect the regional distribution of their host and have potential as a high-resolution tool to trace C. parvum IIaA15G2R1 subtype.

Novel methods for the molecular discrimination of Fasciola spp. on the basis of nuclear protein-coding genes.

Fasciolosis is an economically important disease of livestock caused by Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola flukes. The aspermic Fasciola flukes have been discriminated morphologically from the two other species by the absence of sperm in their seminal vesicles. To date, the molecular discrimination of F. hepatica and F. gigantica has relied on the nucleotide sequences of the internal transcribed spacer 1 (ITS1) region. However, ITS1 genotypes of aspermic Fasciola flukes cannot be clearly differentiated from those of F. hepatica and F. gigantica. Therefore, more precise and robust methods are required to discriminate Fasciola spp. In this study, we developed PCR restriction fragment length polymorphism and multiplex PCR methods to discriminate F. hepatica, F. gigantica, and aspermic Fasciola flukes on the basis of the nuclear protein-coding genes, phosphoenolpyruvate carboxykinase and DNA polymerase delta, which are single locus genes in most eukaryotes. All aspermic Fasciola flukes used in this study had mixed fragment pattern of F. hepatica and F. gigantica for both of these genes, suggesting that the flukes are descended through hybridization between the two species. These molecular methods will facilitate the identification of F. hepatica, F. gigantica, and aspermic Fasciola flukes, and will also prove useful in etiological studies of fasciolosis.

Molecular characterization of Cryptosporidium parvum detected in Japanese black and Holstein calves in Iwate Prefecture and Tanegashima Island, Kagoshima Prefecture, Japan.

Cryptosporidium oocysts were found in 43 out of 77 calves from two farms in Iwate Prefecture and nine farms on Tanegashima Island, Kagoshima Prefecture, Japan. The DNA fragments of 18S ribosomal RNA (18S rRNA) gene were amplified by a nested PCR from 43 oocyst-positive as well as one oocyst-negative samples. All of them were precisely identified as C. parvum by analyzing the nucleotide sequences of the 18S rRNA gene. C. parvum oocyst-positive calves ranged in age from 6 to 13 days old and significantly have watery diarrhea (P<0.05). Sequences of the gene encoding the 60-kDa glycoprotein (GP60) in 43 Cryptosporidium oocyst-positive samples were identical to that of the zoonotic IIaA15G2R1 subtype. We therefore suggest that calves could be potential sources of C. parvum infections in humans.

Molecular characterization of Cryptosporidium parvum from two different Japanese prefectures, Okinawa and Hokkaido.

Infectious diarrhea is the most frequent cause of morbidity and mortality in neonatal calves. Cryptosporidium parvum is one of the main pathogens associated with calf diarrhea. Although diarrhea is a symptom of infection with various pathogens, investigations to detect the types of pathogens have never been performed in Japan. This study investigated the prevalence of four major diarrhea-causing pathogens in calves: C. parvum, rotavirus, coronavirus, and enterotoxigenic Escherichia coli (E. coli K99). Commercial immunochromatography testing of all four pathogens and molecular analysis of C. parvum with diarrhea in calves from southernmost Okinawa and northernmost Hokkaido, Japan, were conducted. The frequencies of C. parvum, rotavirus, coronavirus, and E. coli (K99) in Okinawa were 50%, 28%, 2.3%, and 4.7%, respectively. Watery fecal stools were significantly correlated with C. parvum (p<0.05). In oocyst calculations for C. parvum, no significant difference was observed between the single-infection cases and the mixed-infection cases with rotavirus. Interestingly, molecular analyses targeting small subunit ribosomal RNA as well as glycoprotein 60 (GP60) genes revealed that the C. parvum nucleotide sequences from the two prefectures were identical, indicating that C. parvum with a uniform characteristic is distributed throughout Japan. GP60 subtyping analysis identified C. parvum from Okinawa and Hokkaido as belonging to the IIaA15G2R1 subtype, a known zoonotic subtype. Hence, control of cryptosporidiosis is important not only for pre-weaned calves, but also for human health.