Genetic analyses of Chinese isolates of Toxoplasma gondii reveal a new genotype with high virulence to murine hosts.

A great deal of evidence demonstrates that a strongly clonal population structure of Toxoplasma gondii strains exists in humans and animals in North America and Europe, while the strains from South America are genetically separate and more diverse. Potential differences in virulence between different strains mean that an understanding of strain diversity is important to human and animal health. However, to date, only one predominant genotype, ToxoDB#9 (Chinese I), and a few other genotypes, including ToxoDB#205, have been identified in China. By using DNA sequence-based phylogenetic analyses, we have re-evaluated the population structure of T. gondii strains collected from China and compared them with other global strains. Based on phylogenetic analysis of restriction fragment length polymorphisms, multilocus sequence typing and intron sequences from T. gondii, we propose that the Chinese isolates described as Chinese I are divided into two groups called Chinese I and Chinese III. Our results demonstrate that significant differences were found in mouse mortality caused by some Chinese strains, and also the archetypal I, II, III strains in mice. Furthermore, a comparison of cyst loading in the brains of infected rats showed some Chinese strains to be capable of a high degree of cyst formation. Furthermore we show that genotyping using neutral genetic markers may not be a useful predictor of pathogenic phenotypes.

Prevalence of Toxoplasma gondii in localized populations of Apodemus sylvaticus is linked to population genotype not to population location.

Toxoplasma gondii is a globally distributed parasite infecting humans and warm-blooded animals. Although many surveys have been conducted for T. gondii infection in mammals, little is known about the detailed distribution in localized natural populations. In this study, host genotype and spatial location were investigated in relation to T. gondii infection. Wood mice (Apodemus sylvaticus) were collected from 4 sampling sites within a localized peri-aquatic woodland ecosystem. Mice were genotyped using standard A. sylvaticus microsatellite markers and T. gondii was detected using 4 specific PCR-based markers: SAG1, SAG2, SAG3 and GRA6 directly from infected tissue. Of 126 wood mice collected, 44 samples were positive giving an infection rate of 34.92% (95% CI: 27.14-43.59%). Juvenile, young adults and adults were infected at a similar prevalence, respectively, 7/17 (41.18%), 27/65 (41.54%) and 10/44 (22.72%) with no significant age-prevalence effect (P = 0.23). Results of genetic analysis of the mice showed that the collection consists of 4 genetically distinct populations. There was a significant difference in T. gondii prevalence in the different genotypically derived mouse populations (P = 0.035) but not between geographically defined populations (P = 0.29). These data point to either a host genetic/family influence on parasite infection or to parasite vertical transmission.

A molecular and ecological analysis of the trematode Plagiorchis elegans in the wood mouse Apodemus sylvaticus from a periaquatic ecosystem in the UK.

The prevalence of the digenean Plagiorchis sp. was investigated in a natural wood mouse population (Apodemus sylvaticus) in a periaquatic environment. Classical identification was complemented with the use of molecular differentiation to determine prevalence and verify species identity. Use of the complete ITS1-5.8S rDNA-ITS2 and partial 28S rDNA gene sequences have confirmed that the species reported at this location was Plagiorchis elegans and not Plagiorchis muris as reported previously. This underlines the difficulties in identification of these morphologically similar parasites. Plagiorchis elegans is typically a gastrointestinal parasite of avian species but has also been reported from small mammal populations. Although the occurrence of this digenean in A. sylvaticus in the UK is rare, in the area immediately surrounding Malham Tarn, Yorkshire, it had a high prevalence (23%) and a mean worm burden of 26.6 ± 61.5. The distribution of P. elegans followed a typically overdispersed pattern and both mouse age-group and sex were determined to be two main factors associated with prevalence. Male mice harboured the majority of worms, carrying 688 of 717 recovered during the study, and had a higher prevalence of 32.4% in comparison to only 8.7% in the small intestine of female mice. A higher prevalence of 43% was also observed in adult mice compared to 14% for young adults. No infection was observed in juvenile mice. These significant differences are likely to be due to differences in the foraging behaviour between the sexes and age cohorts of wood mice.

Development and evaluation of loop-mediated isothermal amplification (LAMP) for rapid detection of Clonorchis sinensis from its first intermediate hosts, freshwater snails.

Clonorchiasis, caused by Clonorchis sinensis, is a key foodborne zoonosis, which is mainly found in China, Korea and Vietnam. Detection of this parasite from the second intermediate host, the freshwater fish is the common method for epidemiological surveys of this parasite, but is time consuming, labour intensive and easily leads to misdiagnosis. In this study, we have developed a rapid, sensitive and reliable molecular method for the diagnosis of C. sinensis from its first intermediate hosts, freshwater snails, based on a loop-mediated isothermal amplification (LAMP) method. The specific amplified fragment from genomic DNA of C. sinensis did not cross-react with those from other relevant trematodes and a range of hosts (freshwater fish, shrimps and snails) of C. sinensis living in similar environments. The detection limit of the LAMP method was as low as 10 fg which was 1000 times more sensitive than conventional PCR, which was also demonstrated by successful application to field samples. These results show that the LAMP method is a more sensitive tool than conventional PCR for the detection of C. sinensis infection in the first intermediate hosts and, due to a simpler protocol, is an ideal molecular method for field-based epidemiological surveys of this parasite.

Identification of a new species of digenean Notocotylus malhamensis n. sp. (Digenea: Notocotylidae) from the bank vole (Myodes glareolus) and the field vole (Microtus agrestis).

Notocotylus malhamensis n. sp. is described from the caecum of the bank vole (Myodes glareolus) and the field vole (Microtus agrestis) from Malham Tarn Nature Reserve in North Yorkshire, UK. In total, 581 specimens were collected from rodents trapped at a wetland site (Tarn Fen) between July 2010 and October 2011 with a prevalence of 66·7% and mean intensity of 94·6 in the bank vole and 50% prevalence and a mean intensity of 4·3 in the field vole. This species appears to be most closely related to other previously described Notocotylus species infecting rodents in Europe but differs principally by the metraterm to cirrus sac ratio (1:1·5-1:1·2) in combination with a densely spinulated cirrus, simple caeca and a greater number of ventral glands in the lateral rows (14-17). The use of molecular differentiation was of limited use in this study due to a paucity of relevant information in the DNA sequence databases. However, the complete ITS1-5.8S rDNA-ITS2 and partial 28S gene sequences have been generated to provide a definitive tool for identification of this species in future studies. As far as we know this is the first report of a notocotylid infection in M. glareolus in the UK.

Lipoma or liposarcoma? A cautionary case report.

MERRF syndrome (myoclonic epilepsy with ragged-red fibres) is a mitochondrial encephalomyopathy characterised by a mixed seizure disorder and myoclonus. The condition is associated with multiple large cervical lipomas that often require surgical excision. Comprehensive clinical examination combined with pre-operative radiographic imaging is vital in such cases to help differentiate benign fatty masses from potential liposarcomas. We describe a case in which a lipoma-like liposarcoma was identified following excision of what was expected, on clinical and radiological grounds, to be a lipoma. This case highlights the potential for sarcomatous changes in suspicious lipomas, and the key role that imaging plays in differentiating benign from malignant.

Toxoplasma gondii infection in pregnant women in China.

Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is one of the most common parasitic infections in humans. Primary infection in pregnant women can be transmitted to the fetus leading to miscarriage or congenital toxoplasmosis. Carefully designed nationwide seroprevalence surveys and case-control studies of risk factors conducted primarily in Europe and America, have shaped our view of the global status of maternal and congenital infection, directing approaches to disease prevention. However, despite encompassing 1 in 5 of the world's population, information is limited on the status of toxoplasmosis in China, partly due to the linguistic inaccessibility of the Chinese literature to the global scientific community. By selection and analysis of studies and data, reported within the last 2 decades in China, this review summarizes and renders accessible a large body of Chinese and other literature and aims to estimate the seroprevalence in Chinese pregnant women. It also reviews the prevalence trends, risk factors, and clinical manifestations. The key findings are (1) the majority of studies show that the overall seroprevalence in Chinese pregnant women is less than 10%, considerably lower than a recently published global analysis; and (2) the few available appropriate studies on maternal acute infection suggested an incidence of 0·3% which is broadly comparable to studies from other countries.

Prevalence and co-infection of Toxoplasma gondii and Neospora caninum in Apodemus sylvaticus in an area relatively free of cats.

The protozoan parasite Toxoplasma gondii is prevalent worldwide and can infect a remarkably wide range of hosts despite felids being the only definitive host. As cats play a major role in transmission to secondary mammalian hosts, the interaction between cats and these hosts should be a major factor determining final prevalence in the secondary host. This study investigates the prevalence of T. gondii in a natural population of Apodemus sylvaticus collected from an area with low cat density (<2·5 cats/km2). A surprisingly high prevalence of 40·78% (95% CI: 34·07%-47·79%) was observed despite this. A comparable level of prevalence was observed in a previously published study using the same approaches where a prevalence of 59% (95% CI: 50·13%-67·87%) was observed in a natural population of Mus domesticus from an area with high cat density (>500 cats/km2). Detection of infected foetuses from pregnant dams in both populations suggests that congenital transmission may enable persistence of infection in the absence of cats. The prevalences of the related parasite, Neospora caninum were found to be low in both populations (A. sylvaticus: 3·39% (95% CI: 0·12%-6·66%); M. domesticus: 3·08% (95% CI: 0·11%-6·05%)). These results suggest that cat density may have a lower than expected effect on final prevalence in these ecosystems.

Genotyping of Toxoplasma gondii isolates from cats in different geographic regions of China.

Fourteen isolates of Toxoplasma gondii were isolated from cats from 4 different geographic provinces (Anhui, Hubei, Shanxi and Guangdong) in China and their genetic diversity with 8 nuclear loci SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico, was analysed by restriction fragment length polymorphisms (RFLPs). Two genotypes from these 14 isolates were identified but none of them belongs to the typical genetic types (types I, II and III). It is unexpected that such high similarity was observed in these 14 isolates although their original regions are significantly distant. Our results strongly indicate that the three traditional clonal lineages of types I, II and III of this parasite may not be preponderant in China. In addition, our results show that the genotypes of T. gondii in China may be highly clonal with atypical genotypes and higher virulence.

Evidence for high levels of vertical transmission in Toxoplasma gondii.

Toxoplasma gondii is a highly ubiquitous and prevalent parasite. Despite the cat being the only definitive host, it is found in almost all geographical areas and warm blooded animals. Three routes of transmission are recognised: ingestion of oocysts shed by the cat, carnivory and congenital transmission. In natural populations, it is difficult to establish the relative importance of these routes. This paper reviews recent work in our laboratory which suggests that congenital transmission may be much more important than previously thought. Using PCR detection of the parasite, studies in sheep show that congenital transmission may occur in as many as 66% of pregnancies. Furthermore, in families of sheep on the same farm, exposed to the same sources of oocysts, significant divergent prevalences of Toxoplasma infection and abortion are found between different families. The data suggest that breeding from infected ewes increases the risk of subsequent abortion and infection in lambs. Congenital transmission rates in a natural population of mice were found to be 75%. Interestingly, congenital transmission rates in humans were measured at 19.8%. The results presented in these studies differ from those of other published studies and suggest that vertical transmission may be much more important than previously thought.

Molecular epidemiology of African sleeping sickness.

Human sleeping sickness in Africa, caused by Trypanosoma brucei spp. raises a number of questions. Despite the widespread distribution of the tsetse vectors and animal trypanosomiasis, human disease is only found in discrete foci which periodically give rise to epidemics followed by periods of endemicity A key to unravelling this puzzle is a detailed knowledge of the aetiological agents responsible for different patterns of disease--knowledge that is difficult to achieve using traditional microscopy. The science of molecular epidemiology has developed a range of tools which have enabled us to accurately identify taxonomic groups at all levels (species, subspecies, populations, strains and isolates). Using these tools, we can now investigate the genetic interactions within and between populations of Trypanosoma brucei and gain an understanding of the distinction between human- and nonhuman-infective subspecies. In this review, we discuss the development of these tools, their advantages and disadvantages and describe how they have been used to understand parasite genetic diversity, the origin of epidemics, the role of reservoir hosts and the population structure. Using the specific case of T.b. rhodesiense in Uganda, we illustrate how molecular epidemiology has enabled us to construct a more detailed understanding of the origins, generation and dynamics of sleeping sickness epidemics.

Neospora caninum: detection in wild rabbits and investigation of co-infection with Toxoplasma gondii by PCR analysis.

Neospora caninum is an important pathogen of cattle causing significant economic loss. There is much current interest in wild animal reservoirs for this parasite. The role of the rabbit in this is currently unknown. DNA samples from the brains of wild rabbits (Oryctolagus cuniculus) collected from the Malham area of the Yorkshire dales were investigated by species-specific PCR for the presence of N. caninum and Toxoplasma gondii. We found prevalences of N. caninum of 10.5% (6/57) and T. gondii of 68.4% (39/57) with 8.8% (5/57) co-infected. Strain typing of T. gondii positive rabbits revealed strain types I-III were present in this population. Investigation of tissue distribution determined N. caninum DNA was most often detected in the brain and heart, less often in the tongue and not in the liver. To our knowledge this is the first report of N. caninum detection in naturally infected wild rabbits.

Evidence that primary infection of Charollais sheep with Toxoplasma gondii may not prevent foetal infection and abortion in subsequent lambings.

A study carried out on a sheep farm examined whether Toxoplasma gondii foetal infection and associated abortion occur in successive lambings. We identified 29 ewes that gave birth to lambs on at least 2 successive years over our study period, 2000-2003. Tissue samples from the progeny of these ewes were analysed by PCR to determine infection status with T. gondii. T. gondii-infected lambs were born in 31% of successive pregnancies. T. gondii-positive lambs were aborted in successive pregnancies in 21% of lambings during study period, 2000-2003. The frequency of successive abortions within this flock over the period 1992-2003 was 18%. If a lamb was congenitally infected there was a high risk (69%) that the successive lamb from that ewe would also be congenitally infected. Similarly, if a lamb was aborted there was a high risk (55%) of abortion in the next lamb produced. These data suggest that life-long immunity to T. gondii infections may not always be acquired following an initial infection and raises the question as to whether the mechanisms of T. gondii transmission prior to and during ovine pregnancies are fully understood.

Back pain in osteoporotic vertebral fractures.

UNLABELLED: This review article examines the epidemiology and pathogenesis of back pain and vertebral fractures in osteoporosis, reviewing the management of pain in patients with vertebral fractures and the direct and indirect effect of osteoporosis treatments on back pain. INTRODUCTION: The management of patients with vertebral fractures has largely concentrated on the prevention of further fractures by the treatment of underlying osteoporosis, with drug treatment for acute and chronic back pain and the non-pharmacological management of vertebral fractures receiving less attention. DISCUSSION: Emerging evidence suggests that, in addition to reducing the incidence of vertebral fractures, calcitonin, intravenous bisphosphonates and teriparatide may also have a direct effect on bone pain. Targeted analgesia, tailored to individual need is often required in both the acute and chronic phases following vertebral fracture. Vertebroplasty and kyphoplasty have also been approved for use in the management of vertebral fractures and may prove useful in selected patients unresponsive to conventional pain relief. There is some evidence to support the use of individualised tailored exercise programmes aimed at strengthening back muscles to maintain bone density and reduce further fracture incidence. In addition the use of specific orthoses may help to reduce kyphosis, improve mobility and reduce pain. CONCLUSION: Chronic back pain associated with vertebral fracture provides a great challenge to health care professionals and the patient. This demands a combination of options, including not only therapeutic interventions, but also physiotherapy, psychological support and patient education.

The occurrence of the trematode Plagiorchis muris in the wood mouse Apodemus sylvaticus in North Yorkshire, UK.

Wood mice Apodemus sylvaticus were trapped each September over a 13-year period, from 1993 to 2005, in a wooded area adjacent to Malham Tarn, Yorkshire, UK. Plagiorchis muris was found to be the dominant intestinal trematode and occurred in every year of sampling, with an overall prevalence of 16.9%. This appears to be the first record of P. muris in A. sylvaticus within the UK. The mean worm burden was 2.03 and the distribution of the parasite within mice was typically overdispersed. No difference in prevalence relative to host sex was evident although there was a higher prevalence of 21.9% in larger older mice compared with 9.1% in juveniles, which probably relates to a greater foraging activity in older mice. Annual prevalence values of P. muris infections varied significantly over the study period with higher prevalences being associated with years with a high spring/summer rainfall. The second intermediate hosts for P. muris include a variety of aquatic insect larvae and it is likely that the higher rainfall may result in the occurrence of temporary water bodies suitable for the development of insect larvae.

Does vertical transmission contribute to the prevalence of toxoplasmosis?

Toxoplasma gondii is a ubiquitous parasite with a widespread distribution both in terms of geographical and host range. Although the definitive host is the cat, it is also a major health hazard to domestic animals and humans. Three routes of transmission are recognised (infection from the cat, carnivory and congenital transmission). We aimed to assess the relative importance of congenital transmission, using sheep as a model system, due to the lack of carnivory. We report, using PCR as a diagnostic tool, that congenital transmission occurs with high frequency (69%). If transmission from oocysts was important in sheep, we would expect sheep reared under the same environmental conditions (i.e. a single farm) to have a random distribution of Toxoplasma infection. Using breeding records in conjunction with PCR, some families were found to have high Toxoplasma prevalence and abortion while others were free of Toxoplasma infection and abortion (P < 0.01). This supports the notion that Toxoplasma may be transmitted vertically. In humans, we conducted a similar study and showed that Toxoplasma was transmitted from mother to baby in 19.8% of cases. Vertical transmission in Toxoplasma may be more important than previously thought and this knowledge should be considered in any eradication strategies.

Comparison of musculoskeletal ultrasound practices of a rheumatologist and a radiologist.

OBJECTIVE: There is considerable debate regarding the role of the rheumatologist ultrasonographer and how this development will impact on musculoskeletal ultrasound (MSUS) performed by radiologists. We compared the MSUS practices of a rheumatologist and a radiologist working within the same National Health Service Trust. METHODS: A retrospective review of MSUS reports of consecutive scans performed by a consultant rheumatologist with a special interest in MSUS and a consultant musculoskeletal radiologist. Reports were analysed for referring specialties, indications for MSUS, joint regions scanned, MSUS findings, frequency with which patients were referred for injection and how often injection was performed. RESULTS: A total of 170 patients were referred to the rheumatologist for MSUS of 282 joint regions (91% referred by rheumatologists). Of those, 84 (49%) patients had MSUS examination of more than one joint region, with up to five regions scanned per sitting. One hundred patients were referred to the radiologist for MSUS of 111 joint regions (49% referred by orthopaedic surgeons). The most frequently requested primary indication for MSUS performed by the rheumatologist was detection of synovitis [74 (44%) patients] while MSUS performed by the radiologist was most frequently for assessment for major structural changes [44 (44%) patients]. The rheumatologist performed MSUS-guided injection in 59 of 170 (35%) patients scanned and the radiologist in 13 of 100 (13%). CONCLUSION: MSUS performed by the rheumatologist was predominantly requested by rheumatologists to aid diagnosis of synovial and tendon inflammation and to guide injections, while MSUS performed by the radiologist was predominantly requested by orthopaedic surgeons to aid diagnosis of structural pathology. Curriculums in MSUS designed for rheumatologists may need to place appropriate emphasis on the identification of synovial and tendon inflammation, and injection guidance.

The prevalence of Neospora caninum and co-infection with Toxoplasma gondii by PCR analysis in naturally occurring mammal populations.

Neospora caninum and Toxoplasma gondii are closely related intracellular protozoan parasites associated with bovine and ovine abortion respectively. Little is known about the extent of Neospora/Toxoplasma co-infection in naturally infected populations of animals. Using nested PCR techniques, based on primers from the Nc5 region of N. caninum and SAG1 for T. gondii, the prevalence of N. caninum and its co-infection with T. gondii were investigated in populations of Mus domesticus, Rattus norvegicus and aborted lambs (Ovis aries). A low frequency of infection with N. caninum was detected in the Mus domesticus (3%) and Rattus norvegicus (4.4%) populations. A relatively high frequency of infection with N. caninum was detected in the brains of aborted lambs (18.9%). There was no significant relationship between N. caninum and T. gondii co-infection. Investigation of the tissue distribution of Neospora, in aborted lambs, showed that Neospora could not be detected in tissues other than brain and this was in contrast to Toxoplasma where the parasite could be frequently detected in a range of tissues.