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Doi:10.1016/j.tvjl.2008.03.025

Available online at www.sciencedirect.com The Veterinary Journal xxx (2008) xxx–xxx Detection of Ehrlichia canis by polymerase chain reaction Nuno Alexandre a,*, Ana Sofia Santos b, Maria Sofia Nu´ncio b, Rita de Sousa b, a Departamento de Zootecnia, Universidade de E´vora, E´vora, Portugal b Centro de Estudos de Vectores e Doencßas Infecciosas, Instituto Nacional de Sau´de Dr. Ricardo Jorge, Edifı´cio LEMES, Lisboa, Portugal c Departamento de Sanidade Animal, Faculdade de Medicina Veterina´ria, Universidade Te´cnica de Lisboa, Lisboa, Portugal Antibodies against Ehrlichia canis, the cause of canine monocytic ehrlichiosis, have been reported previously in clinically ill and stray dogs from Portugal. In this study, the 16S rRNA gene of E. canis was detected by the polymerase chain reaction (PCR) in 12/55 (22%) ofdogs with suspected tick-borne disease in the Algarve region in Portugal.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Ehrlichia canis; Polymerase chain reaction; Canine; Portugal Canine monocytic ehrlichiosis (CME) is a tick-borne aged 3 months to 10 years, exhibiting clinical signs of tick- disease caused by Ehrlichia canis, an intracellular bacte- borne disease that were presented to one hospital and seven rium transmitted by the brown dog tick Rhipicephalus san- veterinary clinics in the Algarve region from February to CME include fever, depression, anorexia, weight loss, epi- An automated cell counter (Hemavet 850, CDC Tech- staxis and ocular, gastrointestinal and respiratory signs nologies) was used to perform a complete cell count on blood samples collected from affected dogs into ethylene anaemia and hypergammaglobulinaemia are the main lab- diamine tetraacetic acid. After leucocyte concentration, buffy coat smears were stained with Diff-Quik (Medion The indirect immunofluorescence assay (IFA) has been Diagnostics GmbH) and examined under 1000Â magnifi- widely used to investigate canine ehrlichiosis in Portugal.
Antibodies against E. canis were detected by IFA in 27/ the buffy coat (Flexigene DNA Kit, Qiagen GmbH) in par- 61 (44%) stray dogs from southern Portugal ( However, the IFA may cross-react with different Nested PCR was performed using the outer primers species of Ehrlichia or microorganisms from other closely ECC-ECB and inner primers HE3-CANIS derived from related genera. To confirm that E. canis is the agent causing canine ehrlichiosis in Portugal, the polymerase chain reac- tive samples, two additional hemi-nested PCR reactions tion (PCR) for the 16S rRNA gene was performed on DNA were performed using the universal outer primers EC12- extracted from the blood of 55 dogs (26 males, 29 females), EC9 and inner primers EC10 and EC11 ). PCR amplifications were carried out in a total volume of 50 lL containing 1 lM of each primer, 2.5 U Corresponding author. Tel.: +351 266760829; fax: +351 266760841.
of Taq polymerase, 20 mM of each dNTP, 10 mM 1090-0233/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tvjl.2008.03.025 Please cite this article in press as: Alexandre, N., et al., Detection of Ehrlichia canis by polymerase chain reaction ., The VeterinaryJournal (2008), doi:10.1016/j.tvjl.2008.03.025 N. Alexandre et al. / The Veterinary Journal xxx (2008) xxx–xxx Tris–HCL, 1.5 mM MgCl2 and 50 mM KCl (Master Taq tion ). In this study, E. canis infection was Kit, Eppendorf). Ten microlitres of buffy coat DNA detected by nested PCR in 7/12 dogs that were seronegative extract were used for the initial amplification and 1 lL of by indirect IFA results, reinforcing the value of molecular PCR product in nested reactions. Amplification conditions techniques in the early diagnosis of CME. PCR should be used in conjunction with serology for diagnosis of CME (E. canis Oklahoma strain DNA) and negative samples.
Previously, evidence of E. canis infection in Portugal DNA amplicons from positive samples were purified (Jet- was based on serology ). In the present study, quick Purification Kit, Genomed GmbH) and sequenced we have amplified and sequenced the 16S rRNA gene of using an ABI automated sequencer (Applied Biosystems).
E. canis, providing the first molecular confirmation that Sequence homology searches in GenBank were performed this agent is involved in canine disease in Portugal.
Antibodies against E. canis were detected by IFA None of the authors of this paper has a financial or per- (kindly provided by J.E. Dawson, CDC) as the antigen sonal relationship with other people or organisations that source. Plasma diluted 1:160 in phosphate buffered saline could inappropriately influence or bias the content of the was incubated for 30 min on antigen coated slides and bound antibodies were detected after a second incubationwith fluorescein isothiocyanate-conjugated anti-dog immu- noglobulin G (IgG) (Sigma Immunochemicals). A con-firmed case of CME was defined by a positive PCR result This research was partially supported by the Portuguese with or without an IFA positive result. Probable CME government through the Fundacßa˜o para a Cieˆncia e a cases were IFA positive but PCR negative.
Tecnologia Grant BD/8610/2002. We thank to all veteri- CME was confirmed by PCR in 12/55 (22%) dogs (8 narians directly involved in sample collection. Also, we males, 4 females) with suspected tick-borne disease. Nucle- would like to thank the staff of the Unidade Laboratorial otide sequences of the 16S rRNA gene from all 12 positive de Utilizacßa˜o Comum, Instituto Nacional de Saude Dr. Ri- dogs were identical. Nested PCR with species-specific prim- cardo Jorge for assistance with sequencing.
ers produced a 334 base pair (bp) fragment from the 50 endof the 16S rRNA gene (GenBank EF051166). Hemi-nested reactions with universal primers generated a 684 bp frag-ment from the 30 end of the 16S rRNA gene (EU491504).
Anderson, B.E., Dawson, J.E., Jones, D.C., Wilson, K.H., 1991. Ehrlichia These sequences had 100% identity with E. canis strains chaffeensis, a new species associated with human ehrlichiosis. Journal from dogs in Turkey (AY621071), Taiwan (EU143637), of Clinical Microbiology 29, 2838–2842.
Davoust, B., Boni, M., Parzy, D., 1999. Apport du laboratoire au (AF373613) and Greece (EF011111), 99.9% identity (334/ diagnostic de l’ehrlichiose monocytaire canine. Revue Francßaise desLaboratoire 310, 25–32.
334 and 666/667) with a Spanish strain (AY394465) and Dawson, J.E., Biggie, K.L., Warner, C.K., Cookson, K., Jenkins, S., 99.7% identity (333/334 and 577/579) with an Israeli strain Levine, J.F., Olson, J.G., 1996. Polymerase chain reaction evidence of (U26740). Partial 16S rRNA gene sequences of the Portu- Ehrlichia chaffeensis, an etiologic agent of human ehrlichiosis, in dogs guese isolates had a nucleotide deletion at position 876 rel- from Southeast Virginia. American Journal of Veterinary Research 57, ative to the E. canis prototype strain Jake (CP000107).
Groves, M.G., Dennis, G.L., Amyx, H.L., Huxsoll, D.L., 1975. Trans- IgG against E. canis was detected by IFA in the plasma mission of Ehrlichia canis to dogs by ticks (Rhipicephalus sanguineus).
of 5/12 (41.6%) cases confirmed as CME by PCR. Five American Journal of Veterinary Research 36, 937–940.
other IFA positive dogs had negative PCR results. E. canis Harrus, S., Bark, H., Waner, T., 1997. Canine monocytic ehrlichiosis: an morulae were observed in buffy coat smears of 1/12 PCR update. Compendium on Continuing Education for the Practicing positive dogs by direct microscopy and 0/43 PCR negative Neer, T.M., Breitschwerdt, E.B., Greene, R.T., Lappin, M.R., 2002.
dogs. PCR positive dogs exhibited pyrexia (>39 °C; Consensus statement on ehrlichial disease of small animals from the n = 12), anorexia (n = 12), lymphadenopathy (n = 8), epi- infectious disease study group of the ACVIM. Journal of Veterinary staxis (n = 2), icterus (n = 1), petechial haemorrhages on the skin (n = 1) and vomiting (n = 1). Haematological find- Silveira, C.A., 1992. Ehrlichiose canina. Estudo clı´nico de uma populacßa˜o ings included thrombocytopaenia (<200 Â 103/lL; n = 12), animal, na regia˜o urbana e rural de Setu´bal – Implicacßo˜es em sau´depu´blica e sau´de pu´blica veterina´ria. M.Sc., thesis, Faculdade de anaemia (<5.5 Â 106/lL; n = 9) and leucopaenia (<6 Â Medicina Veterina´ria, Universidade Te´cnica de Lisboa, Lisboa, PCR has been widely used in the laboratory diagnosis of Wen, B., Rikihisa, Y., Mott, J.M., Greene, R., Kim, H.Y., Zhi, N., Couto, CME, especially during the acute phase of the disease C.G., Unver, A., Barstch, R., 1997. Comparison of nested-PCR with before antibodies are detectable ). E. canis immunofluorescent-antibody assay for detection of Ehrlichia canisinfection in dogs treated with doxycycline. Journal of Clinical DNA can be detected by nested PCR in the buffy coats of experimentally infected dogs from the fourth day of infec- Please cite this article in press as: Alexandre, N., et al., Detection of Ehrlichia canis by polymerase chain reaction ., The VeterinaryJournal (2008), doi:10.1016/j.tvjl.2008.03.025

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