Multiple-locus variable-number tandem repeat analysis of Leptospira interrogans and Leptospira borgpetersenii isolated from small feral and wild mammals in East Asia.

Leptospira spp. are the causative agents of a worldwide zoonosis, leptospirosis, maintained by various mammals. Each Leptospira serovar is frequently associated with a particular maintenance host, and recently, Leptospira genotype-host association has also been suggested to limit serovars to restricted areas. We investigated the molecular characteristics of L. interrogans and L. borgpetersenii which were isolated from small feral and wild animals in four East Asian states using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA using 11 loci was performed on 110 L. interrogans serogroups from Japan (79 strains of 5 serogroups from 3 animal species), Philippines (21; 3; 2), Taiwan (7; 2; 3), and Vietnam (3; 1; 1). A MLVA method using 4 loci for L. borgpetersenii was established and performed on 52 isolates from Japan (26; 3; 7), Philippines (13; 1; 2), and Taiwan (13; 1; 3). In L. interrogans, serogroups Autumnalis and Hebdomadis appeared more genetically diverse than serogroups Bataviae, Grippotyphosa, Icterohaemorrhagiae, Pomona, or Pyrogenes. The former serogroup strains with the exception of one Hebdomadis strain were isolated from Apodemus speciosus while all the latter serogroup strains with the exception of Grippotyphosa were isolated from Rattus norvegicus. L. borgpetersenii was isolated from at least 11 animal species while L. interrogans was isolated from five species, which might suggest a wider host range for L. borgpetersenii. Broad host preference in a single genotype was also observed, which colonized not only different species of the same genera but also multiple animal genera. This study demonstrates that there may be variability in the range of genetic diversity among different Leptospira serogroups, which may be attributed to maintenance host animals and environmental factors.

Multiple-locus variable-number tandem repeat analysis and clinical characterization of Leptospira interrogans canine isolates.

Canine leptospirosis occurs worldwide; however, information on the relationship between Leptospira serotypes/genotypes and virulence in dogs remains limited. We investigated the molecular characteristics of Leptospira interrogans canine isolates belonging to three serogroups using multiple-locus variable-number tandem repeat analysis (MLVA) and the effects of each serotype/genotype on the clinical characteristics of leptospirosis in dogs. MLVA using 11 loci of the three major L. interrogans serogroups in Japan, Australis (32 strains from 21 dogs), Autumnalis (12; 7) and Hebdomadis (66; 39), revealed more divergent genetic heterogeneity within each serogroup than multilocus sequence typing (MLST), and they formed two, three and five clusters (CLs), respectively. Lethal infections were caused by all Leptospira serogroup isolates (70.3 % with Hebdomadis, 83.3 % with Australis and 100 % with Autumnalis) or Leptospira isolates belonging to all the CLs (57.1-100 %) without any significant differences. A significant difference in hyperaemia and haemorrhage of mucus membrane was observed between serogroups Australis and Autumnalis (P = 0.03). Leptospira isolates of Australis CL2 caused no hyperaemia and haemorrhage from mucus membrane, whereas those of Australis CL1, Autumnalis CL3 and Hebdomadis CL1 and CL3 did (P<0.05). Significant differences in creatinine (Cre) levels were observed between serogroups Australis and Hebdomadis (P = 0.02). In addition, significant differences in blood urea nitrogen levels were observed between serogroups Australis and Hebdomadis (P = 0.004) and Australis and Autumnalis (P = 0.02). Based on MLVA types, a significant difference in Cre levels was observed between Hebdomadis CL1 and CL4 (P = 0.0018). Our results indicated that MLVA had a higher discriminatory power and was more concordant with serotyping than MLST. Although all Leptospira serotypes and genotypes caused lethal infections in dogs, the L. interrogans serogroup Australis strains were more likely to cause severe kidney damage than Autumnalis and Hebdomadis, which may be more critical to the outcome of infected dogs than haemorrhage. Our results also suggest that the virulence mechanisms and target organs in dogs may differ by Leptospira genotype.

Molecular detection of Diphyllobothrium nihonkaiense in humans, China.

The cause of diphyllobothriosis in 5 persons in Harbin and Shanghai, China, during 2008-2011, initially attributed to the tapeworm Diphyllobothrium latum, was confirmed as D. nihonkaiense by using molecular analysis of expelled proglottids. The use of morphologic characteristics alone to identify this organism was inadequate and led to misidentification of the species.

Onchocerca lupi infection in Turkey: a unique case of a rare human parasite.

Onchocerpa lupi was first isolated from a wolf in Russia. Since then, canine ocular onchocercosis has been increasingly reported, particularly in Europe and the United States. It is thought that blackflies and midges are the vectors of transmission, and it is possible that these vectors could transmit the parasite to humans. The first human case of O. lupi in Turkey was reported in 2011. In this report we present the third human case of O. lupi infection in Turkey. Our patient was a 28-year-old male who displayed a painless, immobile mass under the conjunctiva. The mass measured 10 × 12 mm in size. Pathological examination of the surgically excised tissue was suggestive of infection by a filarial nematode. Subsequently, the parasite was identified as O. lupi through molecular analysis. All of the previously reported cases of O. lupi in both humans and dogs were more symptomatic than in our patient, Onchocerca infection should not be ruled out during the differential diagnosis of the subconjunctival and orbital cystic mass in instances where there is little to no inflammation. It is important to consider biopsy and carry out molecular analysis to identify the parasite.

Current status of lung fluke metacercarial infection in freshwater crabs in the Kawane area of Shizuoka Prefecture, Japan.

Feline cases of lung fluke infection were recently reported in the upper basin of the Oi River in Shizuoka Prefecture. The causative species of these cases were not identified, although a field survey conducted about 40 years ago in this area demonstrated the prevalence of Paragonimus miyazakii metacercariae in intermediate host crabs. To clarify the current status of lung fluke metacercarial infection in host crabs, we collected the Japanese freshwater crab or Sawagani, Geothelphusa dehaani, at 14 sampling sites in the Kawane area, which is located in the upper basin of the Oi River. Sawagani harboring Paragonimus metacercariae were collected at all sampling sites in this study with a total prevalence of 42% (281 of 677 crabs examined) and a range of 16% to 92%. The number of metacercariae per infected crab ranged from 1 to 19 with a mean of 2.2. Based on the morphological features of the metacercariae and adult worms recovered from rats that were experimentally infected with the metacercariae, the lung flukes examined were identified as P. miyazakii Kamo, Nishida, Hatsushika and Tomimura, 1961; ITS2 sequence data corroborate this conclusion. P. miyazakii is still widely prevalent in this area, implying that the recently reported feline paragonimiasis cases were likely caused by P. miyazakii infection.

Molecular and serological investigation of Leptospira and leptospirosis in dogs in Japan.

Canine leptospirosis, which is caused by infection with pathogenic Leptospira species, occurs worldwide, but information regarding the causative Leptospira serotypes and genotypes and their effects on virulence in dogs remains limited. Monitoring acute leptospirosis in dogs as sentinels can also aid in estimating the risk of human leptospirosis, particularly when the disease is rare, as it currently is in Japan. Among 283 clinically suspected cases of leptospirosis diagnosed from August 2007 to March 2011 in Japan, 83 cases were laboratory diagnosed as leptospirosis by blood culture, a rise in antibody titres in paired sera using a microscopic agglutination test (MAT) and/or DNA detection using flaB-nested PCR. The infected dogs comprised hunting dogs (31 dogs) and companion animals (50 dogs) and two unknown; 63.4 % of the infected dogs were males. The mortality rate was 53.2 %. A rise of at least fourfold in MAT titre was detected in 30 dogs whose paired serum samples were obtained, and the predominant reactive serogroup was Hebdomadis (53.3 %), followed by Australis (16.7 %) and Autumnalis (16.7 %). Leptospira interrogans was isolated from 45 dogs of the following serogroups: Australis (16), Autumnalis (six), Canicola (one), Hebdomadis (21) and Icterohaemorrhagiae (one). All of these serogroups caused lethal infections (57.1-100 %). Genetic heterogeneity was demonstrated in serogroups Australis, Autumnalis and Hebdomadis by multilocus sequence typing (MLST) and/or RFLP analysis based on PFGE. In serogroup Hebdomadis, each genotype determined by MLST had a unique mortality rate in the infected dogs. Although classic canine leptospirosis is associated with serovars Canicola and Icterohaemorrhagiae, serogroup Hebdomadis has become the predominant serogroup causing high mortality in Japan. This study suggests that the virulence of members of serogroup Hebdomadis in dogs may be associated with the genotypes in this serogroup.

Leptospira infection at the University of Peradeniya Teaching Hospital, Sri Lanka: clinical and laboratory investigations.

To help formulate a local intervention for leptospirosis in Sri Lanka, we determined the serogroups of leptospiral species among 97 patients diagnosed with leptospirosis at the University of Peradeniya Teaching Hospital, Sri Lanka. Ninety-two point eight percent of the patients were men; nearly two-thirds were > or = 35 years old; the majority had secondary or higher education level, half were farmers or laborers; and 57.7% presented in the acute-phase of the illness. Twenty-five patients (25.8%) were confirmed to have leptospirosis by a positive laboratory method; 17 and 8 cases were confirmed with a positive test by quantitative MAT and nested PCR, respectively. Of the 17 MAT positive cases, infection occurred in a variety of serogroups, but the predominant groups were Sejroe and Tarassovi. Of the 8 nested PCR positive cases, 7 were seen among those with a MAT titer <200 and 1 occurred in a patient with a MAT titer > or = 200 but <400. Of the 8 PCR positive cases, 7 were infected with the leptospiral species L. interrogans. Approximately 26% of the clinically diagnosed patients were confirmed by the two laboratory methods. Laboratory positivity was based on the time of blood collection after the onset of fever. Further studies are warranted to refine the clinical diagnostic criteria and to develop more efficient and accurate diagnostic tests for leptospirosis in resource limited settings.

Validity of the bear tapeworm Diphyllobothrium ursi (Cestoda: Diphyllobothriidae) based on morphological and molecular markers.

The bear tapeworm Diphyllobothrium ursi is described based upon the morphology of adult tapeworms recovered from the brown bear (Ursus arctos middendorffi) and larval plerocercoids found in sockeye salmon (Oncorhynchus nerka) from Kodiak Island in Alaska in 1952. However, in 1987 D. ursi was synonymized with Diphyllobothrium dendriticum, and the taxonomic relationship between both species has not subsequently been revised. In this study mitochondrial cytochrome c oxidase subunit 1 gene (cox1) sequences of holotype and paratype D. ursi specimens that had been preserved in a formalin-acetic acid-alcohol solution since the time the species was initially described approximately 60 yr ago were analyzed. Molecular and phylogenetic analysis of the cox1 sequences revealed that D. ursi is more closely related to D. dendriticum than it is to Diphyllobothrium nihonkaiense and Diphyllobothrium latum. In addition to molecular evidence, differences in the life cycle and ecology of the larval plerocercoids between D. ursi and D. dendriticum also suggest that D. ursi is a distinct species, separate from D. dendriticum and D. nihonkaiense, and also possibly from D. latum .

Molecular identification of a causative parasite species using formalin-fixed paraffin embedded (FFPE) tissues of a complicated human pulmonary sparganosis case without decisive clinical diagnosis.

PCR-based molecular diagnosis was made for the identification of causative agents of the clinically suspected pulmonary proliferative sparganosis case found in Thailand using formalin-fixed paraffin-embedded (FFPE) biopsy specimens. As a reference, FFPE biopsy specimen from a typical cutaneous sparganosis case was examined together. DNA samples were extracted from tissues and two partial fragments of cytochrome c oxidase subunit 1 (cox1) gene were amplified for the detection of Spirometra DNA. Two cox1 fragments were amplified successfully for both specimens. After alignment of nucleotide sequences of the PCR-amplicons, the causative agents of both cases were identified as Spirometra erinaceieuropaei.

Detection of Anaplasma phagocytophilum from Wild Boars and Deer in Japan.

This study was conducted to determine the prevalence and geographic distribution of Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, in wild deer and boars in Japan. We analyzed the blood samples of the study animals using PCR-targeting the 16S rDNA followed by DNA sequencing. Wild deer infected with Anaplasma spp., including Anaplasma bovis and Anaplasma centrale were detected in the region from Hokkaido to Kyushu. The infection rates of A. phagocytophilum, A. bovis, and A. centrale in deer were 15.6, 21.9, and 37.5%, respectively, and the corresponding infection rates in wild boar were 3.6, 17.9, and 3.6%, respectively. However, p44/msp2 genes specific to A. phagocytophilum were not detected among the 16S rDNA-positive samples on PCR analysis. In addition, the 16S rDNA sequences of A. phagocytophilum were 100% similar to those detected previously in the deer in Japan and 98.6% similar to those of A. phagocytophilum detected in the United States and Europe, and from the tick, Ixodes ovatus and Ixodes persulcatus in Japan. These findings suggested that the A. phagocytophilum-related sequences detected from deer and wild boars in Japan were different from those of typical A. phagocytophilum strains.

Prevalence and carrier status of leptospirosis in smallholder dairy cattle and peridomestic rodents in Kandy, Sri Lanka.

Leptospirosis is an important bacterial zoonotic disease globally and one of the notifiable diseases in Sri Lanka. Other than human leptospirosis, little information is available on leptospirosis in domestic and feral animals in Sri Lanka. Thus, this study attempted to determine the prevalence and carrier status of leptospirosis in smallholder dairy cattle and peridomestic rodents to understand the impact of the disease on public health in Kandy, Sri Lanka. Cattle and rodent samples were collected from the Yatinuwara and Udunuwara divisional secretaries in Kandy. Serum samples were analyzed for the presence of antileptospiral antibodies using microscopic agglutination test. DNA was extracted from cattle urine and rodent kidney tissue samples, in which polymerase chain reaction was carried out to detect the Leptospira flaB gene. The cattle in 19 (38.8%) of the 49 farms harbored antileptospiral antibodies. Out of 113 cattle serum samples, 23 (20.3%) were positive; 17 (73.9%) and 6 (26.1%) reacted with serogroups Sejroe and Hebdomadis, respectively. Out of the 74 rodent samples, 13 (17.5%) were positive; 8 (61.5%) and 4 (30.8%) had reactions to serogroups Javanica and Icterohaemorrhagiae, respectively. Leptospiral DNA was detected in one cattle urine sample and identified as Leptospira interrogans. This study revealed a high prevalence of leptospirosis in cattle and rodents in Kandy. These animals were infected with a wide array of leptospiral serogroups, which are consistent with the research findings observed in humans in Kandy. Overall, serological data indicate that relative to rodents, cattle may be a more significant reservoir for human transmission and a greater source of potential risk to local agricultural communities.

Serologic and molecular studies of Leptospira and leptospirosis among rats in the Philippines.

Rats are known to be the most important reservoirs and transmission sources of leptospirosis. However, the status of leptospirosis in the Philippines regarding reservoirs and transmission remains unknown. A survey was conducted in Metro Manila and Laguna that analyzed samples obtained from 106 rats. Using the microscopic agglutination test, we found that 92% of rat serum samples were positive for anti-Leptospira antibodies; the most common infecting serovars were Manilae, Hebdomadis, and Losbanos. On the basis of pulsed-field gel electrophoresis and gyrase B gene sequence analyses, four groups of rat kidney isolates were found: L. interrogans serovar Manilae, serovar Losbanos, and serogroup Grippotyphosa, and L. borgpetersenii serogroup Javanica. Most isolates were lethal after experimental infection of golden Syrian hamsters. Results showed that these four Leptospira serovars and serogroups are circulating among rats, and that these animals may be one of the possible transmission sources of leptospirosis in the Philippines.

Serological and genetic analysis of leptospirosis in patients with acute febrile illness in kandy, sri lanka.

Leptospirosis has emerged as an important infectious disease in Sri Lanka and little information is available on circulating leptospiral species and serogroups in this country. Therefore, we studied circulating leptospiral species and serogroups in patients with acute febrile illness using polymerase chain reaction and the microscopic agglutination test, respectively. Anti-leptospiral antibodies were detected in 26 of 107 serum samples studied (24.3%). The predominant reacting serogroups were Sejroe (9/26, 34.6%) and Icterohaemorrhagiae (5/26, 19.2%). Leptospiral DNA was detected in 3 of the 107 serum samples. The deduced leptospiral species were Leptospira interrogans and L. kirschneri (2 and 1 samples, respectively). These results confirm the existence of a wide array of leptospiral species and serogroups in Sri Lanka and would help to thoroughly elucidate the epidemiology of leptospirosis in this country.

Prevalence of Leptospira spp. in the kidneys of wild boars and deer in Japan.

We surveyed the prevalence of Leptospira spp. from 2005 to 2008 in wild boars and deer in Japan using polymerase chain reaction. Leptospiral flaB was detected in the kidneys of wild boars (positive ratio, 15.2%; 22 of 145) from 9 prefectures and a deer (1.1%; 1 of 94) from 1 prefecture in Japan. There was no annual change in the prevalence of positive animals during the investigation period (chi-squared test, p=0.94) or in the prevalence in male and female wild boars in the 2007 to 2008 season (Fisher's exact test, P=0.45). The Leptospira species harbored by these animals were deduced to be L. interrogans (from 22 animals) and L. borgpetersenii (from 1 animal).

Human leptospirosis cases and the prevalence of rats harbouring Leptospira interrogans in urban areas of Tokyo, Japan.

Thirteen patients with leptospirosis were identified, as confirmed by laboratory analysis during the last 5 years in our laboratory, who came from urban areas of Tokyo, Japan. All of the patients came into contact with rats before the onset of illness. Seventeen per cent of Norway rats captured in the inner cities of Tokyo carried leptospires in their kidneys. Most of these rat isolates were Leptospira interrogans serovar Copenhageni/Icterohaemorrhagiae. Antibodies against these serovars and their DNA were detected in the patients. This suggests that rats are important reservoirs of leptospirosis, and that rat-borne leptospires occur in urban areas of Tokyo.

Isolation and characterization of Leptospira spp. from raccoons in Japan.

We investigated whether raccoons (Procyon lotor) carried leptospires in their kidneys in Japan. Leptospira was isolated from 2 of 71 raccoons captured in Kanagawa Prefecture and 1 of 53 raccoons at a zoological park in Nagasaki Prefecture. Anti-Leptospira antibodies were detected in 16 of 124 raccoons (12.9%) in Kanagawa and 33 of 53 raccoons (62.3%) in Nagasaki, respectively. The partial nucleotide sequences of their flaB genes suggested that the isolates belonged to L. interrogans. The serovars of the isolates were identified as Copenhageni/Icterohaemorrhagiae (1 strain in Kanagawa) and Hebdomadis (1 strain both in Kanagawa and Nagasaki) by reactivity with the reference antisera and restriction fragment length polymorphism (RFLP) analysis based on pulsed-field gel electrophoresis and cross-agglutination-absorption test, respectively. RFLP analysis on the serovars Hebdomadis strains revealed genetic diversity among serovar Hebdomadis. Although it is unclear if the raccoons carried leptospires in their kidneys at the time imported, there is no doubt that imported animals are a new reservoir animal of leptospires in Japan.

Investigation of reservoir animals of Leptospira in the northern part of Miyazaki Prefecture.

We surveyed reservoir animals of leptospires in the northern part of Miyazaki Prefecture, where a cluster of human leptospirosis had occurred during the summer of 2006. Leptospira was isolated from 6 of 57 large Japanese field mice (Apodemus speciosus). The serogroups of the isolates were Autumnalis (5 strains) and Hebdomadis (1 strain) and the partial nucleotide sequences of their flaB genes suggested that the isolates belonged to L. interrogans. The human patient sera reacted specifically with the Leptospira strain isolated from the mice captured around the area where each patient occurred, suggesting that mice are the source of human infection. We also detected leptospiral DNAs by flaB-polymerase chain reaction in the kidneys of large feral animals; wild boars (positive ratio 10.3%; 4 of 39) and deer (19.2%; 10 of 52). The Leptospira spp. harbored by these animals were deduced to be L. interrogans (in 5 animals) and L. borgpetersenii (in 9 animals) by the nucleotide sequences of the amplicons. Anti-Leptospira antibodies were also detected among symptomatic hound dogs. These results suggest that these feral animals may cause leptospirosis and pose a potential risk to hunters and workers in the meat processing industry.