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World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 VALIDATION OF A DIAGNOSTIC METHOD FOR THE DETECTION AND QUANTIFICATION OF BLUETONGUE VIRUS IN ADULT CULICOIDES BITING MIDGES (DIPTERA: CERATOPOGONIDAE). *Eva Veronesi , Peter P.C. Mertens, Philip S. Mellor and Simon Carpenter Institute for Animal Health, Ash Road, Pirbright, Surrey, GU24 ONF, UK. Introduction: Bluetongue (BT) is a non-contagious arthropod-borne viral disease of domesticated and wild ruminants that is transmitted by the bite of adult females, from certain species of Culicoides biting midge (Diptera: Ceratopogonidae). Screening of field caught midges for the presence of bluetongue virus (BTV) can provide valuable information concerning BTV transmission during an outbreak of the disease. In this study we describe the validation of an efficient method to quantify BTV from adult midges. We present results for virus isolation from adult C. sonorensis (derived from the colony at IAH Pirbright) that were intrathoracically infected with BTV, then homogenised using a conventional mortar and pestle method. These data were compared with results obtained using a TissueLyser® machine (Qiagen), which homogenises samples by high frequency shaking in the presence of a ball-bearing. This machine has already been used in several laboratories to extract viral DNA or RNA from infected tissues or insects (e.g. to isolate West Nile Virus from wild caught mosquitoes) but has not previously been validated for quantification of virus in an arboviral/vector system. A quantitative assessment of viral load in the vector is thought to be particularly important for BTV, since a titre of ≥3.0 log10TCID50/midge represents a fully disseminated infection (Fu et al. 1999) and is required for virus transmission to the ruminant host during a blood meal. Material & Methods: In order to ensure the presence of the virus, adult C. sonorensis were individually inoculated (intrathoracically) with BTV serotype 9 (BTV-9, IAH reference collection number KOS2001/03). Immediately after inoculation, equal numbers of these insects were homogenised (individually) using either a mortar pestle, or TissueLyser® method. The TissueLyser® homogenisation parameters that were optimised, include: 1) Duration of shaking; 2) Frequency of shaking; 3) Material of the ball-bearing (polyethylene / stainless steel); and 4) Size of ball-bearing (1, 2, 3, 4, 5 mm diameter). The efficiency of virus recovery was assessed by titrating a ten fold dilution series of each homogenised insect on BHK-21 cells. Adult C. sonorensis were also infected via an oral route, then incubated at ±25ºC for 10 days to allow virus replication. Equal numbers of these midges were individually homogenised using either a mortar and pestle or optimised TissueLyser® method, and virus isolation was carried out as before. Real time RT-PCR (Shaw et al 2007) was used to confirm the identity of BTV infected insects. Results: The optimum TissueLyser® homogenization program, involved shaking insects in 100 µl of Glasgow MEM for 1 min at 25 Hz with a 2 or 3 mm stainless steel ball bearing. The virus titres obtained using oral or intrathoracic infection techniques and the optimised program, did not differ significantly from those obtained using a polypropylene motor-driven pestle (a method that is currently in common use for studies of vector competence). Although a significant difference in the amount of virus obtained was observed between different homogenisation programs (H = 57.73; df = 7; P
World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 WILD LIFE AS A SOURCE OF ZOONOTIC DISEASES Tin Tin Myaing and Tay Zar Aye Cho Despite the discovery of cooking last 1.9 million years ago, the risk of zoonotic diseases emerging from hunting and eating wildlife is still of global importance. World landscape and habitat of the wild animals has a kaleidoscope of niches, leading to the diseases transmitted from animals to man. Infectious pathogens of wild animals'origin have become increasingly important on substantial impacts of human health, agricultural production, international trade, wildlife-based economies, native wildlife populations, wildlife conservation, and the health of ecosystems. Expanded demand for bush meat will likely lead to changes in the exposure of humans to potentially zoonotic microbes.The ancient wild life epidemic zoonotic disease was bubonic plague, emerged in 14 th century, killed approximately one third of Europe’s population transmitted by fleas and the reservoir was Ratus ratus. Rabies was described in Mesopotamia in hunting dogs as early as 2,300BC. Alexander the Great passed in 323 BC, died of encephalitis caused by West Nile Virus of a wild bird reservoir as the modern hypotheses suggestion. HIV/AIDs, SARs, Nipah virus, Rabies and Avian Influenza H5N1 are some zoonotic diseases of interest, originated from wild life reservoir. House rat (Rattus rattus robustulus), fruit bats and flying fox (Pteropus vampyrus) and Civet cat(Paguma larvata) were the source of some zoonotic diseases originated from wild animals, populated in Asian countries and Myanmar. There were evidences of warble fly larvae from Asian Elephant (Elephas maximus) transmitted to mahout’s skin at arm and legs, in Myanmar. A 2/50(4%) cases of warble fly transmitted to mahout's skin ( palm and toe) was observed in 2005. Mechanical removal of the embedded larva under the skin had been observed. There was no further systematic treatment as well as no clinical signs except slight itching. Transmission of Anthrax disease 1% (1/100) (cutaneous form) to a worker who cut off the internal organs of the elephant’s dead body was investigated in 2006 but infected person took antibiotic therapy and saved. Tuberculosis infected in persons closed contact with elephants have been observed but not yet been identified. Civet cat (Paguma larvata) was valuable in Myanmar due to its perfume like smell produced from anal gland. Civet cat was said to be the reservoir of SARs disease. Understandings the emergence of zoonotic agents requires knowledge of pathogen biodiversity in wildlife, human-wildlife interactions, anthropogenic pressures on wildlife populations, human behavior and demographic factors. To reduce risk for emerging zoonoses, the public should be educated about the risks associated with wildlife, bush meat, and exotic pet trades, better understanding of how deforestation and associated hunting leads to the emergence of novel zoonotic pathogens. Control of emerging zoonotic diseases of wild life origin should be relies on national, international, regional and cross sectional networks, public health and veterinary services should be more strengthened, especially the services can become more prepared to respond to endemic and epidemic diseases. Veterinarian diagnosticians should have wide knowledge and skillful in their field of interest. On the other hand wild life conservation and animal welfare should also be considered as an important theme. Key words: wild life based economy, emerging zoonoses, bush meat hunting, public health, animal welfare corresponding author: tintinmyaing@mail4u.com.mm Mon 12 November Mon 12 November World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 SURVEILLANCE OF MYCOBACTERIUM BOVIS INFECTION IN CATTLE IN GREAT BRITAIN DURING 2006 K.L.Jahans*, D.R.Worth, J.Brown, J.M.Gunn, M.Okker, E. Wood and L. Potts Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB. Introduction Despite an intensive test and slaughter programme to prevent cattle-to-cattle transmission, Great Britain has one of the highest animal and herd incidences of bovine TB in Europe. The Veterinary Laboratories Agency has seen an increase in isolates submitted for TB diagnosis since 2002 following the occurrence of Foot and Mouth Disease in 2001. During most of that year (mid February to November) routine testing of cattle for bovine tuberculosis was suspended. Up to 2005, the number of incidents in Great Britain has increased by an average of 16% each year since the mid 1980s. Cattle and other bovine species, such as buffaloes and bison, are the natural hosts but nearly all warmblooded animals, including humans can be affected. Species are not all equally susceptible or share the same ability to act as reservoirs of infection for other species: some are considered spill-over hosts, whereas others (e.g. cattle and badgers) act as true maintenance hosts. Materials & methods Bovine tissue samples (approx 20g) were received in sterile plastic pots with a standard form. Samples from other species were also received but tended to be smaller and arrived with a similar form or letter. Details from both submission types were entered on a database. Lesioned tissue was selected over non-lesioned material. Approximately 1cm 3 of tissue containing lesions was placed in 50ml buffered formaldehyde solution for histopathology. The remainder was homogenized with a solution of 5% oxalic acid in order to destroy any contaminating organisms, centrifuged at 1100g for 10 minutes and the resulting deposit washed and resuspended in 0.85% sterile saline. Then approximately 300 µl were sown onto selective media and incubated for 6 weeks at 37 o C. Identification of M. bovis was usually made by its appearance on solid media slopes. PCR and spoligotyping was used in addition for more accurate differentiation using the methods of Wilton and Cousins (1992) and Kamerbeek et al (1997). Molecular typing of Mycobacterium species other than members of the TB complex, M. avium or M. intracellulare was carried out using a kit produced by Hain Lifescience (http://www.hain-lifescience.com). Results In 2006 the TB Diagnosis Section at the Veterinary Laboratories Agency - Weybridge received 13,907 bovine tissue submissions for confirmation of diagnosis by culture. The number of submissions from other species (excluding badgers) was 690 and consisted mainly of a non-random sample of deer, cat, dog, pig, sheep, goat and camelids. 4,710 (34%) of the cattle submissions and 83 (12%) of submissions from these other species were found to be positive for M. bovis. There has been no significant increase in the number of confirmed bovine tuberculosis incidents in Great Britain in 2006 compared to 2005 (2.49% and 2.45% respectively). Discussion & conclusions Spoligotypes have become more widspread and some of this may have been due to the restocking of herds with cattle from areas of high TB prevalence. Veterinarians are gradually making more use of molecular typing data, in conjunction with cattle movement data, as an aid to tracing the origins of infection because spoligotypes are generally found in geographical clusters. The available evidence suggests that cases in farmed and companion animals were the result of infection spill-over from a cattle or wildlife reservoir. References Kamerbeek J, Schouls L ,Kolk A, van Agterveld M, van Soolingen D, Kuijper S, BunschoteA, Molhuizen H, Shaw R, Goyal M and van Embden J (1997). Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. Journal of Clinical Microbiology, Apr 1997, 907-914, Vol 35, No. 4. Wilton S and Cousins D (1992). Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods Appl. 1992 May;1(4):269-73.
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