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Pakistan Vet. J., 2008, 28(1): 17-20.
S. MANZOOR, M. SIDDIQUE, SAJJAD-UR-RAHMAN AND M. ASHRAF1 Department of Microbiology, University of Agriculture, Faisalabad; 1Remount Veterinary School, Sargodha, Pakistan ABSTRACT
Three equine rearing districts of Punjab, Pakistan including Sargodha, Okara and Faisalabad were surveyed. The occurrence of strangles was highest in foals during February to the start of May compared to winter and summer seasons. Strangles-inflicted foals showed inability to eat, strangled breathing with roaring sounds, swollen submandibular and submaxillary lymph nodes, abscessation in the lymph nodes, hyperthermia and anorexia with general debilitating conditions. Morbidity and case fatality rates were 78 and 2%, respectively. Pus samples were collected from 70 foals showing burst lymph node abscesses and processed on blood agar. Typical growth showing pinpoint mucoid colonies, with beta hemolysis and catalase negative characteristics were subjected to API Strep 20 system. The isolates of streptococcus recovered from pus samples include S. equi subspecies equi (54%), S. equi subspecies zooepidemicus (11%), S. dysgalactia subspecies equisimilis (11%) and mixed isolates of S. equisimilis and S. equi (23%). Streptococcus species were found sensitive to Penicillin and Cefotoxime, whereas Gentamycine and Tetracycline were ineffective.
Key words
: Strangles, foals, SDS-PAGE, M-protein, Streptococcus species.
affected foals were investigated to verify the previously With the development of mechanical transport, the number of equines is decreasing in large cities of MATERIALS AND METHODS
Pakistan but its importance is unequivocal in rural and northern areas of the country. In many parts of the Incidence of strangles
Pakistan, donkeys, horses and mules are the only means Three equine rearing Districts of Punjab, Pakistan of traveling in the remote areas (Ashraf, 2001). Equine including Sargodha, Okara and Faisalabad were population in the country was reported as 4.6 million surveyed throughout the year during January to heads, out of which 2.47 millions existed in districts December, 2005, each month, for new cases of Sargodha, Okara and Faisalabad of Punjab strangles in horses and mules of ages from 9 months to (Anonymous, 2006). 2 years (foals) and 2-5 years (adults). New cases of In equines, strangles is a disease commonly strangles during each month were included in the study associated with fever, depression, anorexia (Sweeney et al., 1989; Dalgleish et al., 1993; Wallace et al., 1995), purulent nasal discharge and abscessation of the lymph Collection of pus samples
nodes of the head and neck (George et al., 1983). From January to May 2005, a total of 70 foals (9 Mortality and morbidity associated with this disease months to 2 years of age) with clinical signs of swollen have been reported to be 3.6 and 62%, respectively in submandibular lymph nodes and ruptured abscesses an outbreak in a standard bred farm in Kentucky, USA were approached in the equine rearing districts of Punjab including Sargodha, Okara and Faisalabad. Pus According to Gillespie and Timoney (1981), in samples were collected in sterilized disposable plastic strangles the involvement of S. equisimilis was not syringes and transported in ice packs to the laboratory significant as equine pathogen, whereas Grant et al. (1993) concluded that some horses were infected with both S. equi and S. equisimilis. The incidence of Bacterial isolation and identification
strangles in young equine population distributed in the Each pus sample was cultured separately on thickly populated areas of Punjab has been described in sodium azide blood agar containing 5% defibrinized this paper. Moreover, pus samples from strangles sheep blood and 0.2% sodium azide and incubated at Pakistan Vet. J., 2008, 28(1): 17-20.
370C for 24 hours under anaerobic conditions. Pure Antibiotic sensitivity
growth of typical mucoid beta hemolytic colonies were Streptococcal species were subjected to susceptibility analyzed through Gram’s staining, under microscope studies against eight antibiotics including Penicillin G, and were confirmed for streptococcus genus through Streptomycin, Sulphamethoxazole, Gentamycine, catalase test. Mucoid, beta hemolytic, and catalase Chloramphenicol, Cefotoxime, Tetracycline and negative colonies containing Gram positive cocci were Ampicillin using standard antibiotic discs and dispenser declared as pure streptococci and were further (Bioanalyse® France). For this purpose standard processed through API Strep 20 system (Bio Meriux®, technique elaborated by Cappuccino and Sherman France) for streptococci species confirmation. (2005) was followed. Biochemical tests including Voges Proskauer test, Hipurate test, Esculine test, Pyrolidonyl test, Alpha galactosidae test, Beta glucuronidase test, Beta galactosidase test, Alkaline phosphates test, Leucine The incidence of strangles and occurrence of amino peptidase test, Arginin Dihydrolase test and streptococcus species in strangles-inflicted foals of 9 fermentation of sugars including D-ribose, L-arabinose, months to 2 years of age during four seasons around the D-manitol, D-sorbitol, D-lactose, D-trehalose, Inuline, year was recorded and it was found to be the highest D-raffinose, Starch and Glycogen in API strep 20 kit during spring season (Mid of January to Start of May). system were used to confirm the streptococcal species. Animals from 2 years to 5 years of age showed no clinical signs in any season of the year (Table 1). Molecular comparison between indigenous
Out of 70 pus samples, typical Streptococcus Streptococcus equi and foreign strain CF32
species were recovered from 55 samples (78.57%), 6 M-protein of Streptococcus equi was procured samples had non-streptococcus isolates (8.57%), following the standard method of Woolcock (1974) and whereas 9 samples (12.85%) were declared as sterile was subjected to Sodium Dodecyl Sulphate Poly where no growth appeared even after 96 hours. Single Acrylamide Gel Electrophoresis (SDS-PAGE, Timoney isolate of S. equi was recovered in 54%, followed by and Strickland, 1984). Band pattern observed though mixed isolates of S. equi and S. equisimilis (23%), SDS-PAGE was compared with that of CF32 strain of single isolates of S. equisimilis (11%) and single isolate Streptococcus equi already recovered (Galan and of S. zooepidemicus (11%, Table 2). Molecular analysis of M-protein extracted from S. equi revealed a pattern with bands 30 Kda, 41 Kda, 46 Optimization of modified Todd-Hewitt broth
Kda and 60 Kda. An equal count (4x1011/ml) was Filtered and heat inactivated (560C) horse serum recorded after 24 hours in test tubes containing was dispensed in duplicate at a rate of 2, 5 and 10% in modified Todd-Hewitt broth with 5 and 10% horse three test tubes containing 10 ml of modified Todd- serum at 370C. In broth with 2% horse serum, count Hewitt broth. Fresh culture of S. equi was inoculated in each of three test tubes. These tubes were incubated at 370C and count of streptococcal cells was conducted Each of the three streptococcal species were cultured and growth after 24 hours was subjected to Table 1: Incidence of strangles during January to December 2005 in Punjab, Pakistan
Adult horses/mules (2 – 5 years)
Young foals (9 months-2 years)
(Cases in number)
(Cases in million)
Pakistan Vet. J., 2008, 28(1): 17-20.
Table 2: Streptococcal species isolated from pus samples in strangles cases of foals
No. (%) of
Fermentation test
Ribose Trehalose Sorbitol Lactose
eight antibiotic culture sensitivity discs. From the effective vaccine against strangles. Optimization of results, it is clear that each of the three recovered already modified Todd-Hewitt broth was conducted by streptococcal species was sensitive to Penicillin G and supplementing the broth with different concentrations Cefotoxime and showed intermediate behavior against of horse serum. From the experimentation, it was streptomycin and Sulphamethoxazole. Streptococcal concluded that modified Todd-Hewitt broth species showed resistance against Gentamycine and supplemented with 5% horse serum is economical and efficient compared to broth dispensed with 2% and 10% horse serum for the mass scale growth of S. equi. DISCUSSION
Antibiotic susceptibility of each of three streptococcal species revealed that in vitro Penicillin G A survey for the incidence of strangles in young as and Cefotoxime were very effective against well as adult population of equines round the year was Streptococci. As far as the effects of antibiotics conducted to get an idea about the season in which this treatment in strangles is concerned, it is a different story disease can affect animals and to confirm the age group because in early stages of infection the use of these usually inflicted with this disease. Incidence of antibiotics aborts the reaction and no immunity is strangles was found highest in foals of 9 months to 2 developed and as treatment is withdrawn, the infection years of age during spring season (Mid of January to re-occurs (Harrington et al., 2002). In later stages, lack of sufficient vascularity at the site of infection and Out of total samples, 23% streptococcal isolates abscessation interferes with the antibiotic to combat contained mixed growth of S. equi and S. equisimili and six streptococcal isolates (11%) contained S. equisimilis In the present study, it was seen that incidence of alone, confirming the fact that horses are infected with strangles continuously increased from January to April both S. equi as well as S. equisimilis (Grant et al., and then it started decreasing. In July, no new case was 1993). M-proteins of locally isolated S. equi after reported after the report of few cases in June. Incidence analysis through SDS-PAGE showed a band pattern similar to that found in S. equi strain CF32 thus of strangles from the end of January to the start of May confirming the fact that S. equi is homogenous through in foals of 9 months to 2 years of age was highest out the world (Galan and Timoney, 1987). (2.56%) compared to the adult equines of age from 2 to From the observations made in this study, it is 5 years, confirming the fact that once an animal is recommended that both S. equi as well as S. equisimilis infected with strangles it attains life long immunity should be considered for the development of a safe and Table 3: Antibiotic sensitivity of Streptococcal isolates recovered from strangles cases of foals
Antibiotic Sensitivity Discs (30 micro grams of antibiotic/disc)
Streptomycin Sulphamet Gentam- Chloramp- Cefotoxi- Tetracycline Ampicillin
S = Sensitive, I = Intermediate, R = Resistant Values in parentheses indicate zone diameter in mm. Pakistan Vet. J., 2008, 28(1): 17-20.
George, J. S., J. S. Reif, R. K. Sheider, C. J. Small, R. A higher occurrence of S. equi was recorded in P. Ellis, S. P. Snyder and A. E. McChesney, 1983. young foals compared to adults. S. equi alone and in Identification of carriers of Streptococcus equi in a conjunction with S. equisimilis were isolated in the naturally infected herd. J. Amer.Vet. Med. Assoc., laboratory. The isolates showed maximum sensitivity to penicillin and cefotoxime. Therefore, it may be Grant, S. T., A. Efstratiou and N. Chanter, 1993. recommended to prepare a bivalent vaccine for the Laboratory diagnosis of strangles and the isolation of atypical Streptococcus equi. Vet. Rec., 133: 215- Harrington, D. J., I. C. Sutcliffe and N. Chanter, 2002. REFERENCES
The molecular basis of Streptococcus equi infection and disease. Microbes and Infection, 4: Anonymous, 2006. Economic Survey. Economic Affairs Division, Govt. Pakistan, Islamabad. Sweeney, C. R., C. E. Bensen, W. H. Robert, D. A. Ashraf, M., 2001 Some blood parameters of equines in Meirs, S. O. Birmingham, S. C. Whitehead and D. strangles. PhD Thesis, Univ. Agri, Faisalabad, Cohen, 1989. Description of an epizootic and persistence of Streptococcus equi infection in horses. J. Amer. Vet. Med. Assoc., 194(9): 1281- Microbiology-A laboratory Manual. 6th Ed., Pearson Education (Singapore), Indian branch, Timoney, J. F. and K. L. Strickland, 1984. Lysogeny and immunologically reactive proteins of Dalgleish, R., S. Love, H. M. Pirie, M. Pirie and D. J. Streptococcus equi. Vet. Rec., 115: 148-149. Walker, J. A. and J. F. Timoney, 2002. Construction of Taylor, 1993. An outbreak of strangles in young a stable non-mucoid deletion mutant of the Streptococcus equi Pinnacle vaccine strain. Vet. Galan, J. E. G. and J. F. Timoney, 1987. Molecular analysis of the M-protein of Streptococcus equi and Wallace, F. J., D. Emery, A. W. Cripps and A. J. cloning and expression of the M-protein gene in Escherichia coli. Infec. Immunity, 55(12): 3181- immunization in protection against Streptococcus equi (strangles) infection in horses. Vet. Immunol. Gillespie, J. H. and J. F. Timoney, 1981. Hagen and Bruner’s Infectious Diseases of Domestic Animals. Woolcock, J. B., 1974. Purification and antigenecity of 7th Ed., Cornell Univ. Press, Ithaca, New York, an M-like protein of Streptococcus equi. Inf.



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