Muhammad Ijaz
Epidemiology Diagnosis And Chemotherpy Of Strangles In Equines - 2010
Strangles is an infectious malady of equidae characterized by upper respiratory tract infection, dysponea, anorexia, regional suppurative lymphadenitis and causes high morbidity and low mortality. Considering the significance and utilization of equines in our country and the substantial losses rendered by Strangles, the present project was designed to study epidemiology, diagnosis and chemotherapy of strangles in Lahore and Sargodha districts of the Punjab province in Pakistan.
The present study comprised of five phases. In phase-I, epidemiology of the disease including prevalence, variations in SeM, SzPSe and Se18.9 proteins and mortality rate were studied in Lahore and Sargodha districts. For epidemiology, nasal swabs and pus samples from the affected lymph nodes of 500 equines (nr=250 horses, rutz250 mules) suspected for strangles were collected and cultured for identification of S. equl. The collected samples were processed at Medicine and Microbiology Laboratories of the University of Veterinary and Animal Sciences, Lhore, Pakistan and Gluck equine research center, Department of Veterinary Science, University of Kentucky, USA. Out of 250 horses and 250 mules, 113(45.2%) horses and 99 (3 9.6%) mules tested positive for S. equi. on the basis of culture. Number of S. equl isolates were significantly higher (P<0.05) in pus samples taken from sub-mandibular lymph nodes as compared to nasal discharge samples. The difference was significant (P<0.05) among mules of different age groups. The highest prevalence of strangles
was recorded in horses and mules less than 2 year of age as compared to those having age more than 2 years.
In the present study, prevalence of strangles round the year in horses and mules were also calculated and it was found to be the highest during the months of
February, March, April and May while few cases were seen during the months of January, June and July and no cases were observed during others months. The significant difference was observed (p
the prevalence rate on the basis of PCR and culture of nasal and pus samples from affected submandibular lymph nodes it revealed that the sensitivity of Polymerase
chain reaction appears to be much greater than culture. The culture along with PCR is the best diagnostic technique for S. equi as PCR test does not differentiate between dead and live bacteria, hence a positive test may not correlate with active infection; therefore, a positive culture may be necessary to confirm the diagnosis.
In this phase of epidemiological study of disease, effect of selective pressure of allelic diversity in SeM of S. equi on immunoreactive proteins SzPSe and Se 18.9 was also studied. The aim of this study was to determine whether variations in SeM are accompanied by variations in the immunoreactive surface of exposed SzPSe and secreted Se18.9. Sequences of genes of 25 S. equi alleles isolated from different countries of the world over a period of 40 years were compared. Twenty different SeM alleles were identified including 6 not included in the data base (http:// pubmlst.org/szooepidemicus). Amino acid variation was also detected distal to the N- terminus of SeM. No variation was observed in SzPSe except for an Australian isolate which showed a deletion of one PEPK repeat. The Se 18.9 protein in all 25 isolates of S. equi did not exhibit any variation. Interestingly, only 2 SNP loci were detected in Se 18.9 compared to 93 and 49 in SeM and SzPSe respectively. The greater frequency of mutation in SzPSe compared to Se18.9 may be related to a high rate of recombination of SzPSe and the inclusion of exogenous DNA sequence based on the atypical GC percentage of its central hyper variable region.
In horses the mortality rate was recorded as 1.64% whereas the mortality rate in mules having less than 5 years of age was found to be 0.88%. No significant difference (P>0.05) in mortality rate among horses and mules of different age groups affected with strangles was observed.
In phase-I! of the present study, carrier status of the horses and mules were
studied. Out of 122 horses found positive to PCR, 20 horses (10<2 years and 10 between 2 and 5 years of age) were selected and monitored for 12 weeks. Their nasal
swab samples were used for identification of bacteria through culture and PCR on weekly basis. Till the end of 3rd week all horses <2 years of age remained positive but at the end of 4th to 7th weeks there remained positive only 5, 2, 1 and zero horses out of 10, respectively on the basis of culture whereas through PCR at the end of the 4th week all horse <2 years of age were found positive, but at the end of 5th to 10th weeks there remained 7, 5, 4, 2, 1 and zero horses out of 10, respectively. While all the horses aging between 2 to 5 year, were positive up to the 1St week but at the end of 2nd to 8th week out of 10 there were 9, 7, 6, 3, 1, 1 and zero horses respectively positive on the basis of culture but through PCR, all horses were positive till 4th week but at the end of 5th to 9th week number was reduced to 9, 7, 6, 3, 2 and zero. Similarly, out of 113 mules, 20 mules (10<2 year and 10 between 2 and 5 years of old) were also monitored for 12 weeks to study their carrier status. After the end of 2nd week all mules <2 years of age were positive but at the end of 3rd to 6th weeks there remained 7, 3, 1 and zero mules out of 10, respectively on the basis of culture but through PCR at the end of the 5th week all mules <2 years of age were positive, but at the end of 6th to 10th weeks there remained 9, 7, 3, 2 and zero mules out of 10, respectively. While in 2 and 5 year old mules, all were positive up to the 2nd week but at the end of 3rd to 7th weeks there were 6, 4, 2, 1, 1 and zero mules out of 10, respectively on the basis of culture but through PCR, all mules were positive up to 5th week but at the end of 6th to 10th weeks there were 8, 5, 2, 1 and zero. Horses and mules were declared free of infection on the basis of three consecutive negative samples through culture and PCR.
From the result of present study, it may be concluded that sensitivity of Polymerase Chain Reaction appears to be much greater than culture for study of carrier status of equines. Moreover, recovered animals should be kept in quarantine period at least upto 9th week because the recovered horses and mules remain carrier for prolonged period of time and can act as source of infection for susceptible animals through periodic shedding of S equi. (comprising 10 horses and 10 mules) for in-vivo trials. Efficacy of the antibiotics was assessed weekly on the basis of negative nasal swab culture. Results of in-vitro antibiotic sensitivity revealed that in horses and mules, S equi was most sensitive to Procaine penicillin followed by ceftiofur Na, cephradine, erythromycin, ampicillin, tetracycline, chloramphenicol, sulfamethoxazole, trimethoprim + sulfdiazine and gentamycin whereas the result of in-vivo antibiotic trials revealed that horses and mules suffered from strangles without abscess formation were most sensitive to Procaine penicillin followed by ceftiofur Na, cephradine and erythromycin whereas animals which developed abscess showed no response. It is concluded from the result of present study that Procaine penicillin is most effective in-vitro and in-vivo antibiotic followed by ceftiofur Na and cephradine. These antibiotics might be used for the treatment of strangles infection.
Phase-V, comprised over in-vitro trials of disinfectants. Efficacy of disinfectants, like povidone iodine, 0.6% H2S04, dettol and bleach was assessed. Phenol Co-efficient Test was applied, to ascertain efficacy of these disinfectants, used
in, in-vitro trials. Among four disinfectants, povidone iodine was found to be the best one with a phenol coefficient of 1.25 that is greater than phenol i.e. 1.00 while 0.6%
H2S04 showed similar phenol coefficient as that of phenol. The phenol coefficient of dettol and bleach were observed as 0.5 and 0.75 respectively. Therefore it is recommended that S. equi is highly sensitive to povidone iodine and 0.6% H2S04.
Department of Clinical Medicine & Surgery
Phd. thesis
1211,T
Epidemiology Diagnosis And Chemotherpy Of Strangles In Equines - 2010
Strangles is an infectious malady of equidae characterized by upper respiratory tract infection, dysponea, anorexia, regional suppurative lymphadenitis and causes high morbidity and low mortality. Considering the significance and utilization of equines in our country and the substantial losses rendered by Strangles, the present project was designed to study epidemiology, diagnosis and chemotherapy of strangles in Lahore and Sargodha districts of the Punjab province in Pakistan.
The present study comprised of five phases. In phase-I, epidemiology of the disease including prevalence, variations in SeM, SzPSe and Se18.9 proteins and mortality rate were studied in Lahore and Sargodha districts. For epidemiology, nasal swabs and pus samples from the affected lymph nodes of 500 equines (nr=250 horses, rutz250 mules) suspected for strangles were collected and cultured for identification of S. equl. The collected samples were processed at Medicine and Microbiology Laboratories of the University of Veterinary and Animal Sciences, Lhore, Pakistan and Gluck equine research center, Department of Veterinary Science, University of Kentucky, USA. Out of 250 horses and 250 mules, 113(45.2%) horses and 99 (3 9.6%) mules tested positive for S. equi. on the basis of culture. Number of S. equl isolates were significantly higher (P<0.05) in pus samples taken from sub-mandibular lymph nodes as compared to nasal discharge samples. The difference was significant (P<0.05) among mules of different age groups. The highest prevalence of strangles
was recorded in horses and mules less than 2 year of age as compared to those having age more than 2 years.
In the present study, prevalence of strangles round the year in horses and mules were also calculated and it was found to be the highest during the months of
February, March, April and May while few cases were seen during the months of January, June and July and no cases were observed during others months. The significant difference was observed (p
the prevalence rate on the basis of PCR and culture of nasal and pus samples from affected submandibular lymph nodes it revealed that the sensitivity of Polymerase
chain reaction appears to be much greater than culture. The culture along with PCR is the best diagnostic technique for S. equi as PCR test does not differentiate between dead and live bacteria, hence a positive test may not correlate with active infection; therefore, a positive culture may be necessary to confirm the diagnosis.
In this phase of epidemiological study of disease, effect of selective pressure of allelic diversity in SeM of S. equi on immunoreactive proteins SzPSe and Se 18.9 was also studied. The aim of this study was to determine whether variations in SeM are accompanied by variations in the immunoreactive surface of exposed SzPSe and secreted Se18.9. Sequences of genes of 25 S. equi alleles isolated from different countries of the world over a period of 40 years were compared. Twenty different SeM alleles were identified including 6 not included in the data base (http:// pubmlst.org/szooepidemicus). Amino acid variation was also detected distal to the N- terminus of SeM. No variation was observed in SzPSe except for an Australian isolate which showed a deletion of one PEPK repeat. The Se 18.9 protein in all 25 isolates of S. equi did not exhibit any variation. Interestingly, only 2 SNP loci were detected in Se 18.9 compared to 93 and 49 in SeM and SzPSe respectively. The greater frequency of mutation in SzPSe compared to Se18.9 may be related to a high rate of recombination of SzPSe and the inclusion of exogenous DNA sequence based on the atypical GC percentage of its central hyper variable region.
In horses the mortality rate was recorded as 1.64% whereas the mortality rate in mules having less than 5 years of age was found to be 0.88%. No significant difference (P>0.05) in mortality rate among horses and mules of different age groups affected with strangles was observed.
In phase-I! of the present study, carrier status of the horses and mules were
studied. Out of 122 horses found positive to PCR, 20 horses (10<2 years and 10 between 2 and 5 years of age) were selected and monitored for 12 weeks. Their nasal
swab samples were used for identification of bacteria through culture and PCR on weekly basis. Till the end of 3rd week all horses <2 years of age remained positive but at the end of 4th to 7th weeks there remained positive only 5, 2, 1 and zero horses out of 10, respectively on the basis of culture whereas through PCR at the end of the 4th week all horse <2 years of age were found positive, but at the end of 5th to 10th weeks there remained 7, 5, 4, 2, 1 and zero horses out of 10, respectively. While all the horses aging between 2 to 5 year, were positive up to the 1St week but at the end of 2nd to 8th week out of 10 there were 9, 7, 6, 3, 1, 1 and zero horses respectively positive on the basis of culture but through PCR, all horses were positive till 4th week but at the end of 5th to 9th week number was reduced to 9, 7, 6, 3, 2 and zero. Similarly, out of 113 mules, 20 mules (10<2 year and 10 between 2 and 5 years of old) were also monitored for 12 weeks to study their carrier status. After the end of 2nd week all mules <2 years of age were positive but at the end of 3rd to 6th weeks there remained 7, 3, 1 and zero mules out of 10, respectively on the basis of culture but through PCR at the end of the 5th week all mules <2 years of age were positive, but at the end of 6th to 10th weeks there remained 9, 7, 3, 2 and zero mules out of 10, respectively. While in 2 and 5 year old mules, all were positive up to the 2nd week but at the end of 3rd to 7th weeks there were 6, 4, 2, 1, 1 and zero mules out of 10, respectively on the basis of culture but through PCR, all mules were positive up to 5th week but at the end of 6th to 10th weeks there were 8, 5, 2, 1 and zero. Horses and mules were declared free of infection on the basis of three consecutive negative samples through culture and PCR.
From the result of present study, it may be concluded that sensitivity of Polymerase Chain Reaction appears to be much greater than culture for study of carrier status of equines. Moreover, recovered animals should be kept in quarantine period at least upto 9th week because the recovered horses and mules remain carrier for prolonged period of time and can act as source of infection for susceptible animals through periodic shedding of S equi. (comprising 10 horses and 10 mules) for in-vivo trials. Efficacy of the antibiotics was assessed weekly on the basis of negative nasal swab culture. Results of in-vitro antibiotic sensitivity revealed that in horses and mules, S equi was most sensitive to Procaine penicillin followed by ceftiofur Na, cephradine, erythromycin, ampicillin, tetracycline, chloramphenicol, sulfamethoxazole, trimethoprim + sulfdiazine and gentamycin whereas the result of in-vivo antibiotic trials revealed that horses and mules suffered from strangles without abscess formation were most sensitive to Procaine penicillin followed by ceftiofur Na, cephradine and erythromycin whereas animals which developed abscess showed no response. It is concluded from the result of present study that Procaine penicillin is most effective in-vitro and in-vivo antibiotic followed by ceftiofur Na and cephradine. These antibiotics might be used for the treatment of strangles infection.
Phase-V, comprised over in-vitro trials of disinfectants. Efficacy of disinfectants, like povidone iodine, 0.6% H2S04, dettol and bleach was assessed. Phenol Co-efficient Test was applied, to ascertain efficacy of these disinfectants, used
in, in-vitro trials. Among four disinfectants, povidone iodine was found to be the best one with a phenol coefficient of 1.25 that is greater than phenol i.e. 1.00 while 0.6%
H2S04 showed similar phenol coefficient as that of phenol. The phenol coefficient of dettol and bleach were observed as 0.5 and 0.75 respectively. Therefore it is recommended that S. equi is highly sensitive to povidone iodine and 0.6% H2S04.
Department of Clinical Medicine & Surgery
Phd. thesis
1211,T