Large-scale genome analysis of bovine commensal Escherichia coli reveals that bovine-adapted E. coli lineages are serving as evolutionary sources of the emergence of human intestinal pathogenic strains.

How pathogens evolve their virulence to humans in nature is a scientific issue of great medical and biological importance. Shiga toxin (Stx)-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are the major foodborne pathogens that can cause hemolytic uremic syndrome and infantile diarrhea, respectively. The locus of enterocyte effacement (LEE)-encoded type 3 secretion system (T3SS) is the major virulence determinant of EPEC and is also possessed by major STEC lineages. Cattle are thought to be the primary reservoir of STEC and EPEC. However, genome sequences of bovine commensal E. coli are limited, and the emerging process of STEC and EPEC is largely unknown. Here, we performed a large-scale genomic comparison of bovine commensal E. coli with human commensal and clinical strains, including EPEC and STEC, at a global level. The analyses identified two distinct lineages, in which bovine and human commensal strains are enriched, respectively, and revealed that STEC and EPEC strains have emerged in multiple sublineages of the bovine-associated lineage. In addition to the bovine-associated lineage-specific genes, including fimbriae, capsule, and nutrition utilization genes, specific virulence gene communities have been accumulated in stx- and LEE-positive strains, respectively, with notable overlaps of community members. Functional associations of these genes probably confer benefits to these E. coli strains in inhabiting and/or adapting to the bovine intestinal environment and drive their evolution to highly virulent human pathogens under the bovine-adapted genetic background. Our data highlight the importance of large-scale genome sequencing of animal strains in the studies of zoonotic pathogens.

Retrospective study on the possibility of an SFTS outbreak associated with undiagnosed febrile illness in veterinary professionals and a family with sick dogs in 2003.

INTRODUCTION: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-born disease and its animal-to-human transmission has come to attention recently. During our sero-survey of SFTS virus (SFTSV) among veterinary professionals in 2018, a veterinarian and his assistant working in an animal hospital were tested positive by enzyme-linked immunosorbent assay (ELISA). An additional survey implied a cluster of SFTS cases in which four more people, a family who brought two sick dogs to the animal hospital in 2003, were involved. This study aimed at assessing the possibility of animal-to-human transmission of SFTSV in this cluster. METHODS: Retrospective interviews were performed with the owner family of the dogs and their clinical records were obtained from each hospital. SFTSV-IgG were tested by ELISA and virus neutralization test using the sera collected from them in 2018. RESULTS: The interviews revealed that a total of six people, the two veterinary professionals and the owner family who took care of the sick dogs, suffered from SFTS-like symptoms in the same period of time in 2003. All patients did not have tick bite before the onset and all suspected causative agents were excluded by laboratory tests. The serological tests in this study revealed the four owner family members were all positive for SFTSV antibodies. CONCLUSIONS: Considering the extremely low seroprevalence of SFTSV antibodies among inhabitants of the region, the existence of SFTSV antibodies in all these six people presents a possibility that they were involved in an SFTS outbreak originated in the sick dogs in 2003.

Direct Transmission of Severe Fever with Thrombocytopenia Syndrome Virus from Domestic Cat to Veterinary Personnel.

Two veterinary personnel in Japan were infected with severe fever with thrombocytopenia syndrome virus (SFTSV) while handling a sick cat. Whole-genome sequences of SFTSV isolated from the personnel and the cat were 100% identical. These results identified a nosocomial outbreak of SFTSV infection in an animal hospital without a tick as a vector.

Repair of a calcaneal fracture with a combination of internal and external fixation in a calf.

A 6-day-old male Japanese Black calf presented with a transverse fracture of the left calcaneus. In calcaneal fractures, traction of the gastrocnemius muscle causes substantial displacement of the proximal fracture fragment; therefore, external fixation alone is prone to failure of fusion or deformed fusion. Furthermore, internal fixation alone may result in refracture due to the high load on the implant. Therefore, internal and external fixation were used to treat the fracture. Bone fusion was observed on postoperative day 50; the wire was removed on postoperative day 90. Radiographic examination at 4 months postoperatively revealed that the bone had fused in normal alignment. Therefore, a good prognosis can be expected for calcaneal fractures treated with combined internal and external fixation.

A retrospective analysis for criteria of surgical intervention in Japanese Black calves with respiratory signs caused by perinatal rib fracture.

Medical records of 16 Japanese Black calves with respiratory signs associated with perinatal rib fracture were analyzed, retrospectively, to speculate criteria of surgical intervention for the disease. For this analysis, the severity of respiratory condition was classified into three grades: Grade 1, with no wheezing, Grade 2, with wheezing after excitement but not at rest and Grade 3, with wheezing at rest. Grade 1 (n=3) received only conservative management. Seven of Grade 2 (n=8) and all of Grade 3 (n=5) had surgical management. Clinical outcome was good in all of Grade 1, and the surgical success rate was 83% in other groups. Our result suggests that surgical intervention should be considered for cases with more the severity of Grade 2.

Serological and molecular survey of tick-borne zoonotic pathogens including severe fever with thrombocytopenia syndrome virus in wild boars in Miyazaki Prefecture, Japan.

BACKGROUND: Miyazaki Prefecture is one of the hotspots of severe fever with thrombocytopenia syndrome (SFTS) cases and related deaths in Japan since 2013 and other pathogens of tick-borne diseases (TBDs). Japanese spotted fever and scrub typhus are also endemic in this region. OBJECTIVES: A total of 105 wild boars, hunted in 2009, were serologically examined as sentinels for TBDs to indirectly demonstrate the potential hazard of ticks transmitting pathogens to humans in the studied area. METHODS: The collected blood and spleens of the wild boars underwent serological and molecular tests for SFTSV, Rickettsia japonica (Rj) [antibody to spotted fever group rickettsiae (SFGR) were tested by using species-common antigen], and Orientia tsutsugamushi (Ot). RESULTS: Seroprevalences of SFTSV, SFGR, and Ot were 41.9%, 29.5%, and 33.3%, respectively. SFTS viral RNA was identified in 7.6% of the sera, whereas DNA of Rj or Ot was not detected in any sample. In total, 43.8% of the boars possessed an infection history with SFTSV (viral gene and/or antibody). Of these, 23.8% had multiple-infection history with SFGR and/or Ot. CONCLUSIONS: The high prevalence of SFTSV in wild boars might reflect the high risk of exposure to the virus in the studied areas. In addition, SFTSV infection was significantly correlated with Ot infection, and so were SFGR infection and Ot infection, indicating that these pathogens have common factors for infection or transmission. These data caution of the higher risk of SFTSV infection in areas with reported cases of other TBDs.

Radiographic diagnosis and surgical management for successful outcomes for osteochondrosis in Japanese Black calves.

Osteochondrosis (OC) is not well recognized in cattle. Japanese Black (JB) cattle are valued for their beef and studies regarding OC are scarce. Hence, the aim of this study was to retrospectively discuss clinical and radiographical findings, diagnosis and treatment options for OC in JB cattle for successful outcomes. Medical records of 11 calves affected with OC were reviewed, retrospectively. Clinical and radiographic findings, treatment and outcomes and postmortem examination findings were analyzed. All calves had lameness. Stifle (4/14) and tarsal (4/14) were the most affected joints. Subchondral bone lucencies were observed in all joints (14/14) and accompanied with sclerosis (10/14). Radiographic grading was performed in calves. Calves were treated surgically (8/11) or conservatively (1/11). Surgically treated calves were auctioned (5/8). Postmortem examination was performed (2/11). Gross findings revealed ulcerative lesion at articular surface. Histopathologically, cartilage layer was defected at lesion. Inflammation (2/2) and granulation tissue (1/2) was also observed. In conclusion, OC in JB cattle can be diagnosed on the basis of radiography with history and clinical signs in clinical setting. Surgery should be performed in mild cases for successful outcomes. A modified radiographic grading criteria for clinical cases may hold potential in evaluating prognosis and outcomes.

Pseudorabies virus infection in hunting dogs in Oita, Japan: Report from a prefecture free from Aujeszky's disease in domestic pigs.

We isolated two pseudorabies virus (PRV) isolates (designated OT-1 and OT-2) from two hunting dogs exhibiting neurological manifestations after eating the flesh of wild boar hunted in Oita prefecture, Kyushu Island, Japan. The isolates corresponded to a previously reported PRV (MY-1 strain) isolated from a hunting dog in neighboring Miyazaki prefecture, and it clustered into genotype II based on the glycoprotein C sequence. Our results suggest that this common PRV strain may have been maintained in wild boars on Kyushu Island even though domestic pigs in this area have attained an Aujeszky's disease-free status.

Isolation of Severe Fever with Thrombocytopenia Syndrome Virus from Various Tick Species in Area with Human Severe Fever with Thrombocytopenia Syndrome Cases.

Severe fever with thrombocytopenia syndrome (SFTS), caused by Dabie bandavirus, generally called SFTS virus (SFTSV), is an emerging zoonosis in East Asia. In Japan, 50-100 cases of SFTS have been reported each year since the first case was reported in 2013. SFTS is a tick-borne infectious disease, and SFTSV has been isolated from ticks in China and South Korea. Haemaphysalis longicornis and Amblyomma testudinarium are considered the primary vectors in Japan. However, the other tick species seldom feeding on humans might also play an important role in maintaining the virus in nature. In this study, we collected ticks on vegetation around the location where two SFTS patients were estimated to have been infected in Miyazaki Prefecture, Japan, isolated live SFTSV, and performed a phylogenetic analysis. A total of 257 ticks were collected, and SFTSV RNA was detected in 19.5% (9/46) of tick pools. A total of 10 infectious SFTSVs were successfully isolated from A. testudinarium, Haemaphysalis flava, Haemaphysalis formosensis, Haemaphysalis hystricis, and Haemaphysalis megaspinosa. Furthermore, the whole viral sequences isolated from ticks were highly homologous to sequences isolated from SFTS patients in the same sampling area in the past. These results suggest that SFTSVs are maintained in these tick species in the sampling area and sporadically transmitted to humans. Surveillance of SFTSV in ticks provides important information about the risk of incidental transmission to humans.

Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus in Small-Animal Veterinarians and Nurses in the Japanese Prefecture with the Highest Case Load.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is the causative agent of SFTS, an emerging tick-borne disease in East Asia, and is maintained in enzootic cycles involving ticks and a range of wild animal hosts. Direct transmission of SFTSV from cats and dogs to humans has been identified in Japan, suggesting that veterinarians and veterinary nurses involved in small-animal practice are at occupational risk of SFTSV infection. To characterize this risk, we performed a sero-epidemiological survey in small-animal-practice workers and healthy blood donors in Miyazaki prefecture, which is the prefecture with the highest per capita number of recorded cases of SFTS in Japan. Three small-animal-practice workers were identified as seropositive by ELISA, but one had a negative neutralization-test result and so was finally determined to be seronegative, giving a seropositive rate of 2.2% (2 of 90), which was significantly higher than that in healthy blood donors (0%, 0 of 1000; p < 0.05). The seroprevalence identified here in small-animal-practice workers was slightly higher than that previously reported in other high-risk workers engaged in agriculture and forestry in Japan. Thus, enhancement of small-animal-practice workers' awareness of biosafety at animal hospitals is necessary for control of SFTSV.

Bilateral rostral mandibulectomy for treatment of necrotic mandibular open fractures in two Japanese Black cattle.

Two-month-old (Case 1) and one-year-old (Case 2) Japanese Black cattle presented with bilateral rostral mandibular open fractures. At presentation, the proximal edges of each fracture were discolored and had a severe stench, indicating necrosis caused by infection. In both cases, a bilateral rostral mandibulectomy over the symphysis was performed. Although the tongues of both animals prolapsed post-surgery, they had no significant problems with eating or drinking. Case 1 showed excellent growth during fattening, and Case 2 successfully became pregnant and gave birth. Rostral mandibulectomy can be an effective surgical option for the treatment of cattle with difficulty in internal or external fixation due to unfavorable necrotic cranial mandibular open fractures.

A seroepidemiological survey for paragonimosis among boar-hunting dogs in central and southern Kyushu, Japan.

A seroepidemiological survey for paragonimosis among boar-hunting dogs was conducted throughout central and southern Kyushu, Japan. Among a total of 224 sera collected from boar-hunting dogs kept by 38 owners, 147 (65.6%) were positive for IgG antibodies against Paragonimus westermani antigen by ELISA. In the 147 seropositive dogs, 83 (56.5%) dogs demonstrated high antibody titers (OD>0.700), which appeared to indicate active infection. There were no obvious differences noted in age, sex and the genetic background of the dogs. The seroprevalence was dependent on the feeding manner of the owners and was extremely variable (0-100%). The majority (34/38) of owners fed their dogs uncooked boar meat and/or allowed them free-access to the residues of hunted boars after dissection, resulting in dogs with seropositive results. The dogs that were never fed wild boar meat were entirely seronegative. These results clearly demonstrate that boar-hunting dogs play an important role as a definitive Paragonimus host and that wild boars serve as a reservoir host for the maintenance of the Paragonimus life-cycle, presumably P. westermani, in the mountainous areas of central and southern Kyushu, Japan.

Large-group infection of boar-hunting dogs with Paragonimus westermani in Miyazaki Prefecture, Japan, with special reference to a case of sudden death due to bilateral pneumothorax.

We recently found a group of 5 boar-hunting dogs infected with Paragonimus westermani. As wild boars are known to be the potential paratenic hosts for this parasite, boar-hunting dogs have obviously a high risk of infection by this parasite. In the present study, therefore, we investigated 20 dogs of another group kept by a hunter in Miyazaki Prefecture, Japan, in order to determine whether paragonimosis is a common problem among boar-hunting dogs. The results showed that P. westermani eggs were present in the feces of 10 out of 20 dogs, while 17 dogs were seropositive on ELISA. Taken together with our previous results, it appears that paragonimosis is a serious problem in boar-hunting dogs. The possible risks of infected dogs acting as a source for maintaining the P. westermani life cycle are also discussed.

Infection of a group of boar-hunting dogs with Paragonimus westermani in Miyazaki Prefecture, Japan.

A 5-year-old male mixed-bred boar-hunting dog with a Plott hound background weighing 23 kg was brought to the Veterinary Teaching Hospital, University of Miyazaki, in October 2002. The dog was diagnosed with active infection with the lung fluke Paragonimus westermani by serological testing and also by detection of parasite eggs in his feces. Subsequent examination of four other dogs working with this dog as a boar-hunting team revealed that all five dogs were infected with P. westermani.

A Risk Assessment of Aflatoxin M1 Exposure in Low and Mid-Income Dairy Consumers in Kenya.

Aflatoxin M1 (AFM1), a human carcinogen, is found in milk products and may have potentially severe health impacts on milk consumers. We assessed the risk of cancer and stunting as a result of AFM1 consumption in Nairobi, Kenya, using worst case assumptions of toxicity and data from previous studies. Almost all (99.5%) milk was contaminated with AFM1. Cancer risk caused by AFM1 was lower among consumers purchasing from formal markets (0.003 cases per 100,000) than for low-income consumers (0.006 cases per 100,000) purchasing from informal markets. Overall cancer risk (0.004 cases per 100,000) from AFM1 alone was low. Stunting is multifactorial, but assuming only AFM1 consumption was the determinant, consumption of milk contaminated with AFM1 levels found in this study could contribute to 2.1% of children below three years in middle-income families, and 2.4% in low-income families, being stunted. Overall, 2.7% of children could hypothetically be stunted due to AFM1 exposure from milk. Based on our results AFM1 levels found in milk could contribute to an average of -0.340 height for age z-score reduction in growth. The exposure to AFM1 from milk is 46 ng/day on average, but children bear higher exposure of 3.5 ng/kg bodyweight (bw)/day compared to adults, at 0.8 ng/kg bw/day. Our paper shows that concern over aflatoxins in milk in Nairobi is disproportionate if only risk of cancer is considered, but that the effect on stunting children might be much more significant from a public health perspective; however, there is still insufficient data on the health effects of AFM1.

New hematological key for bovine leukemia virus-infected Japanese Black cattle.

The European Community's (EC) Key, which is also called Bendixen's Key, is a well-established bovine leukemia virus (BLV) diagnostic method that classifies cattle according to the absolute lymphocyte count and age. The EC Key was originally designed for dairy cattle and is not necessarily suitable for Japanese Black (JB) beef cattle. This study revealed the lymphocyte counts in the BLV-free and -infected JB cattle were significantly lower than those in the Holstein cattle. Therefore, applying the EC Key to JB cattle could result in a large number of undetected BLV-infected cattle. Our proposed hematological key, which was designed for JB cattle, improves the detection of BLV-infected cattle by approximately 20%. We believe that this study could help promote BLV control.

Dembo polymerase chain reaction technique for detection of bovine abortion, diarrhea, and respiratory disease complex infectious agents in potential vectors and reservoirs.

Bovine abortion, diarrhea, and respiratory disease complexes, caused by infectious agents, result in high and significant economic losses for the cattle industry. These pathogens are likely transmitted by various vectors and reservoirs including insects, birds, and rodents. However, experimental data supporting this possibility are scarce. We collected 117 samples and screened them for 44 bovine abortive, diarrheal, and respiratory disease complex pathogens by using Dembo polymerase chain reaction (PCR), which is based on TaqMan real-time PCR. Fifty-seven samples were positive for at least one pathogen, including bovine viral diarrhea virus, bovine enterovirus, Salmonella enterica ser. Dublin, Salmonella enterica ser. Typhimurium, and Neospora caninum; some samples were positive for multiple pathogens. Bovine viral diarrhea virus and bovine enterovirus were the most frequently detected pathogens, especially in flies, suggesting an important role of flies in the transmission of these viruses. Additionally, we detected the N. caninum genome from a cockroach sample for the first time. Our data suggest that insects (particularly flies), birds, and rodents are potential vectors and reservoirs of abortion, diarrhea, and respiratory infectious agents, and that they may transmit more than one pathogen at the same time.

Cattle with the BoLA class II DRB3*0902 allele have significantly lower bovine leukemia proviral loads.

The bovine MHC (BoLA) class II DRB3 alleles are associated with polyclonal expansion of lymphocytes caused by bovine leukemia virus (BLV) infection in cattle. To examine whether the DRB3*0902 allele, one of the resistance-associated alleles, is associated with the proviral load, we measured BLV proviral load of BLV-infected cattle and clarified their DRB3 alleles. Fifty-seven animals with DRB3*0902 were identified out of 835 BLV-infected cattle and had significantly lower proviral load (P<0.000001) compared with the rest of the infected animals, in both Japanese Black and Holstein cattle. This result strongly indicates that the BoLA class II DRA/DRB3*0902 molecule plays an important immunological role in suppressing viral replication, resulting in resistance to the disease progression.

Phylogenetic analysis of env gene of bovine leukemia virus strains spread in Miyazaki prefecture, Japan.

To understand how the latest dominant bovine leukemia virus (BLV) strains were introduced and spread in the Miyazaki prefecture, we collected blood samples from 3 geographic areas (north, central and south) and carried out sequence analysis of the BLV env gene. Two genotypes, genotype I, and III, were identified and the majority of the strains belonged to genotype I (71/74). To clarify a route of BLV introduction, we divided the strains into 20 subgenotypes based on their nucleotide sequences and performed phylogenetic analysis. Our study indicated that common BLV strains were comparatively evenly distributed even in the area, where the farmers have not introduced cattle from other areas and the cattle have limited exposure to BLV infection in grazing fields.

Population structure of Escherichia coli O26 : H11 with recent and repeated stx2 acquisition in multiple lineages.

A key virulence factor of enterohaemorrhagic Escherichia coli (EHEC) is the bacteriophage-encoded Shiga toxin (Stx). Stxs are classified into two types, Stx1 and Stx2, and Stx2-producing strains are thought to cause more severe infections than strains producing only Stx1. Although O26 : H11 is the second most prevalent EHEC following O157 : H7, the majority of O26 : H11 strains produce Stx1 alone. However, Stx2-producing O26 strains have increasingly been detected worldwide. Through a large-scale genome analysis, we present a global phylogenetic overview and evolutionary timescale for E. coli O26 : H11. The origin of O26 has been estimated to be 415 years ago. Sequence type 21C1 (ST21C1), one of the two sublineages of ST21, the most predominant O26 : H11 lineage worldwide, emerged 213 years ago from one of the three ST29 sublineages (ST29C2). The other ST21 lineage (ST21C2) emerged 95 years ago from ST21C1. Increases in population size occurred in the late 20th century for all of the O26 lineages, but most remarkably for ST21C2. Analysis of the distribution of stx2-positive strains revealed the recent and repeated acquisition of the stx2 gene in multiple lineages of O26, both in ST21 and ST29. Other major EHEC virulence genes, such as type III secretion system effector genes and plasmid-encoded virulence genes, were well conserved in ST21 compared to ST29. In addition, more antimicrobial-resistance genes have accumulated in the ST21C1 lineage. Although current attention is focused on several highly virulent ST29 clones that have acquired the stx2 gene, there is also a considerable risk that the ST21 lineage could yield highly virulent clones.