A diagnostic study of Echinococcus multilocularis in red foxes (Vulpes vulpes) from Great Britain.

Alveolar echinococcosis is caused by a parasitic tapeworm Echinococcus multilocularis and is a serious disease with high fatality in humans. The definitive primary host is the red fox (Vulpes vulpes) but domestic animals (dogs and to a lesser extent cats) as well as several genera of rodents can also be infected with the parasite. There is, to date, no evidence of indigenous cases of E. multilocularis in Great Britain (GB) but in most of continental Europe the parasite is considered to be endemic and/or slowly spreading. All pet dogs entering the United Kingdom (UK) under the pet travel scheme (PETS) are therefore currently treated with an anthelmintic effective against Echinococcus spp. Surveillance of red foxes is required to demonstrate disease freedom and maintain this regulation to prevent further geographical spread of the parasite to free areas within the EU. A study of 588 wild red foxes collected from across Great Britain (GB) between October 1999 and November 2000 found no Echinococcus spp. This report describes a further study of GB foxes collected predominately during 2005 and 2006. Fox faecal samples (n=384) were examined for both E. multilocularis and Echinococcus granulosus using an egg isolation procedure followed by PCR method, based on published primer sets. A non-specific primer set that amplifies Taenia spp. as well as Mesocestoides, Dipylidium and Diphyllobothrium was also included in the assay to validate the test procedure as these parasites are expected to be more common in wild fox populations. All faecal samples tested negative for both E. multilocularis and E. granulosus but results for approximately 35% of the samples indicated the presence of Taenia spp. or other closely related cestodes. This data contributes to the evidence that suggests that E. multilocularis is not present in mainland Britain and justifies the requirement for ongoing surveillance to demonstrate disease freedom.

Development of a real-time PCR assay for the detection of Trichinella spiralis in situ.

Trichinellosis is a widespread zoonotic disease caused by nematodes of the genus Trichinella. Most human infections are caused by Trichinella spiralis, with pig meat being the main source of infection. As a consequence, all countries in the EU inspect slaughtered animals to prevent the distribution of infected meat to consumers. However, Trichinella spp. infect nearly all orders of mammals and so wildlife monitoring is often required in regions that want to provide evidence of negligible risk of infection in pigs. Surveys of the parasite in the Red fox are generally accepted as evidence of the wildlife reservoir. The EU reference method of detection in food animals for human consumption involves digestion of the host muscle followed by microscopic screening of the resultant sediment for trichinae and the method has been adapted for use with foxes. This work describes the development of a real-time PCR assay for the detection of Trichinella in fox tissue. The assay was designed to the Internal Transcribed Spacer 2 region of the Trichinella genome. Initial assay development was carried out using infected mouse tissue, as positive foxes have not been reported in the UK since 1957. The developed assay, which was shown to be specific for T. spiralis, was then tested using fox muscle spiked with isolated larvae at the rate of 1 larva per gram (LPG) of muscle tissue, as this is the theoretical detection limit using the digest method, as well as 0.5 and 0.1 LPG. The PCR assay was shown to detect the larvae at the higher infection rates and, by testing dilutions of the extracted DNA, it was demonstrated that a potential limit of detection of approx. 0.01 larvae per gram of tissue homogenate may be possible.

The detection of horse and donkey using real-time PCR.

We have developed real-time PCR assays specific for horse and donkey, applicable to the detection of low levels of horse or donkey meat in commercial products. Primers, designed to the mitochondrial cytochrome b gene, were 3' mismatched to closely related and other commercial species. Amplification of non-target species DNA was prevented by truncation of primers at the 5' position, thereby conferring complete specificity. Both assays were highly sensitive and detected the presence of 1pg of donkey template DNA or 25pg of horse template DNA when assessed using dilutions of DNA in water. Model food samples, spiked with horse or donkey muscle and commercial products containing horse, were successfully tested for the presence of horse or donkey, demonstrating the applicability of the assays to food products.

Analysis of venom constituents from the parasitoid wasp Pimpla hypochondriaca and cloning of a cDNA encoding a venom protein.

Venom from Pimpla hypochondriaca, an endoparasitoid of pupae, was size-fractionated using gel filtration chromatography and analysed by SDS-PAGE in the presence and absence of reducing agent. A complex mixture of more than 20 venom constituents was identified which ranged in M(r) between approximately 5 and 100 kDa. Venom from a wide range of size fractions inhibited the motility of larval haemocytes and prevented the formation of cell aggregates when analysed in vitro, indicating that anti-haemocytic activity is mediated by multiple venom components. Sephadex A25 beads injected into the haemocoel of pupae were encapsulated within 24h. This reaction was abolished when the pupae were injected with 30 microg of venom protein, equivalent to one-fifth of a venom sac, 1h prior to implantation of the beads, confirming that venom suppresses encapsulation in pupae. Using random 5' end sequencing of a P. hypochondriaca venom gland cDNA library, we have isolated a cDNA encoding a 25.3 kDa protein containing a signal peptide and having sequence similarity to serine proteases. The N-terminal sequence of six residues from two venom proteins of 28 and 30 kDa was the same and identical to amino acids encoded by the cDNA, confirming that two mass forms of the protein are secreted into the venom sac. The N-terminal sequence of both venom proteins began nine residues towards the C terminus following the predicted signal sequence cleavage site, suggesting that the proteins are proteolytically processed before or during storage in the venom sac. The general applicability of using random 5' sequencing to identify cDNAs encoding secretory products is discussed.

Pirimiphos-methyl resistance in two stored product mites, Acarus siro and Acarus farris, as detected by impregnated paper bioassay and esterase activity assays.

The response to pirimiphos-methyl, in one strain of Acarus farris and two strains of Acarus siro, was assessed using an impregnated filter paper bioassay and by the selection of adults following exposure to pirimiphos-methyl. It was concluded that one of the strains of A. siro was resistant to pirimiphos-methyl and that a major resistance mechanism was involved. The second strain of A. siro gave a response similar to that of a laboratory strain unexposed to organophosphates and was considered to be susceptible. The A. farris strain responded to selection at the ED50 but not at the ED99, and it was concluded that a minor resistance mechanism is present in this strain. Assays of esterase activity were used to attempt to identify the biochemical mechanisms involved in the resistance detected by the bioassays. The A. farris and susceptible A. siro strains showed similar levels of esterase activity but the esterase activity of the resistant A. siro strain was significantly greater. An increase in esterase activity followed selection of both the A. farris strain and the resistant A. siro strain. An acetylcholinesterase assay showed no significant difference between the susceptible and pirimiphos-methyl selected strains of A. siro. The results suggest that esterases are involved in the resistance to pirimiphos-methyl found in A. siro and A. farris but that in A. siro, at least, other mechanisms may also be present.

Exploring indicators of telephone nursing quality.

To explore whether documentation, use of clinical guidelines, and nurse competency are the best indicators of quality telephone nursing, this study examined the relationship between these commonly cited indicators and the characteristics of a telephone nursing call. This study, done at a large health maintenance organization (HMO), found: accompanying symptoms played a major role in telephone nursing assessment; call length was related to documentation process and to number of visits to a health care facility after a call; nurses' interpersonal skills and ability to determine urgency of a call are related to the documentation process but not to outcomes of the call; time of a call is related to disposition; and disposition is related to number of visits after a call.

Correction of rectal prolapse by anterior resection.

During the past three consecutive years low anterior resection with rectopexy has been used to correct complete rectal prolapse in nine patients. There have been no recurrences and only two complications, one a presacral abscess not related to anastomotic malfunction, and the other a fecal fistula in an 81-year-old woman, which resolved spontaneously without colostomy.