Diagnostic Potential of Immunoblot Analysis for Identifying Rabbits Infected with Trichophyton mentagrophytes

The aim of this study was to investigate the diagnostic potential of immunoblot analysis for detecting rabbits naturally infected with Trichophyton mentagrophytes. A protein extract of the dermatophyte T. mentagrophytes was prepared from a fungal mat grown in liquid medium, where the production of keratinases was stimulated by keratin as a nitrogen source. The proteins derived from disrupted T. mentagrophytes were separated by SDS-PAGE and visualized by Western blotting. Twenty-one rabbits infected with T. mentagrophytes and 10 non-dermatophyte exposed rabbits were studied. Eight dominant bands were identified in immunoblots from dermatophyte-infected rabbits, with apparent molecular weights ranging from 20 to 75 kDa. The diagnostic potential of the bands for identifying infected rabbits was evaluated by determining sensitivity, specificity, positive and negative predictive values, and overall accuracy. Bands at 20, 30 and 33 kDa were observed with 100 % specificity and 76.19 % sensitivity and can thus be considered highly accurate in correct identification of the infected animals. Accuracies from 54 to 62 % were found for the other dominant bands. Bands at 42, 45 and 75 kDa with 85.71 to 100 % sensitivity appeared not to be specific for dermatophyte infection. Although the specificity of bands at 26 and 28 kDa was found to be 100 %, their accuracy was only moderate, due to the low level of sensitivity, 9.52 and 23.81 %, respectively

Dijagnostička važnost ELISA testova pri određivanju specifičnih antitijela u mačkama i kunićima inficiranim dermatofitozom

Two indirect ELISA tests developed for the detection of specific IgG in cats and rabbits, infected with M. canis and T. mentagrophytes, respectively, were evaluated and compared. The levels of specific antibodies were determined in sera of 20 cats and 25 rabbits naturally infected with M. canis and T. mentagrophytes, respectively. Infection was confirmed by the results of fungal culture. Blood samples from 12 cats and 17 rabbits, previously unexposed to dermatophytes, served as negative controls. A significant increase in the level of specific antibodies in groups of infected animals was demonstrated. Sensitivity, specificity and predictive values of a positive and a negative test were determined to evaluate the diagnostic potential. ELISA for the detection of specific antibodies in cats infected with M. canis (ELISA-cats test) exhibited 75.0 % of sensitivity at 91.7 % of specificity, whereas the test for the detection of specific antibodies in rabbits, infected with T. mentagrophytes (ELISA-rabbits test) is highly sensitive (96.0 %) and highly specific (94.1 %), confirming its encouraging diagnostic potential. The cross-reactivity of fungal antigens was tested by performing the assays with antigens M. canis, T. mentagrophytes, M. pachydermatis and A. fumigatus. There were no significant indications of cross-reactions in the test T. mentagrophytes-rabbits, whereas strong cross-reaction between dermatophyte antigens was observed in the test M. canis-cats.Razrađena su dva indirektna ELISA testa za utvrđivanje specifičnih IgG u mačkama i kunićima inficiranim M. canis i T. mentagrophytes. Testovi su međusobno uspoređeni i procijenjeni. Utvrđena je razina specifičnih antitijela u serumu dvadesetak mačaka i 25 kunića prirodno inficiranih s M. canis i T. mentagrophytes. Infekcija je potvrđena rezultatima fungalne kulture. Kao negativna kontrola koristili su se uzorci krvi 12 mačaka i 17 kunića koji prethodno nisu bili izloženi dermatofitima. U skupini inficiranih životinja utvrđeno je bitno povećanje razine specifičnih antitijela. Utvrđeni su osjetljivost, specifičnost i očekivane vrijednosti u pozitivnim i negativnim testovima kako bi se procijenio dijagnostički potencijal. ELISA-test (ELISA-cats test) za utvrđivanje specifičnih antitijela u mačaka inficiranih s M. canis pokazao je 75,0 % osjetljivosti i 91,7 % specifičnosti, dok je test za utvrđivanje antitijela u kunića inficiranih s T. mentagrophytes (ELISA-rabbits test) bio vrlo osjetljiv (96 %) i jako specifičan (94,1 %), potvrđujući time njegov znatan dijagnostički potencijal. Unakrsna reaktivnost fungalnih antigena ispitana je provodeći pokuse s antigenima M. canis, T. mentagrophytes, M. pachydermatis i A. fumigatus. U testu T. mentagrophytes-rabbits nije opažena značajna unakrsna reakcija dok je u testu M. canis-cats došlo do snažne unakrsne reakcije između antigena dermatofita

Dijagnostička važnost ELISA testova pri određivanju specifičnih antitijela u mačkama i kunićima inficiranim dermatofitozom

Two indirect ELISA tests developed for the detection of specific IgG in cats and rabbits, infected with M. canis and T. mentagrophytes, respectively, were evaluated and compared. The levels of specific antibodies were determined in sera of 20 cats and 25 rabbits naturally infected with M. canis and T. mentagrophytes, respectively. Infection was confirmed by the results of fungal culture. Blood samples from 12 cats and 17 rabbits, previously unexposed to dermatophytes, served as negative controls. A significant increase in the level of specific antibodies in groups of infected animals was demonstrated. Sensitivity, specificity and predictive values of a positive and a negative test were determined to evaluate the diagnostic potential. ELISA for the detection of specific antibodies in cats infected with M. canis (ELISA-cats test) exhibited 75.0 % of sensitivity at 91.7 % of specificity, whereas the test for the detection of specific antibodies in rabbits, infected with T. mentagrophytes (ELISA-rabbits test) is highly sensitive (96.0 %) and highly specific (94.1 %), confirming its encouraging diagnostic potential. The cross-reactivity of fungal antigens was tested by performing the assays with antigens M. canis, T. mentagrophytes, M. pachydermatis and A. fumigatus. There were no significant indications of cross-reactions in the test T. mentagrophytes-rabbits, whereas strong cross-reaction between dermatophyte antigens was observed in the test M. canis-cats.Razrađena su dva indirektna ELISA testa za utvrđivanje specifičnih IgG u mačkama i kunićima inficiranim M. canis i T. mentagrophytes. Testovi su međusobno uspoređeni i procijenjeni. Utvrđena je razina specifičnih antitijela u serumu dvadesetak mačaka i 25 kunića prirodno inficiranih s M. canis i T. mentagrophytes. Infekcija je potvrđena rezultatima fungalne kulture. Kao negativna kontrola koristili su se uzorci krvi 12 mačaka i 17 kunića koji prethodno nisu bili izloženi dermatofitima. U skupini inficiranih životinja utvrđeno je bitno povećanje razine specifičnih antitijela. Utvrđeni su osjetljivost, specifičnost i očekivane vrijednosti u pozitivnim i negativnim testovima kako bi se procijenio dijagnostički potencijal. ELISA-test (ELISA-cats test) za utvrđivanje specifičnih antitijela u mačaka inficiranih s M. canis pokazao je 75,0 % osjetljivosti i 91,7 % specifičnosti, dok je test za utvrđivanje antitijela u kunića inficiranih s T. mentagrophytes (ELISA-rabbits test) bio vrlo osjetljiv (96 %) i jako specifičan (94,1 %), potvrđujući time njegov znatan dijagnostički potencijal. Unakrsna reaktivnost fungalnih antigena ispitana je provodeći pokuse s antigenima M. canis, T. mentagrophytes, M. pachydermatis i A. fumigatus. U testu T. mentagrophytes-rabbits nije opažena značajna unakrsna reakcija dok je u testu M. canis-cats došlo do snažne unakrsne reakcije između antigena dermatofita

Diagnostic Potential of Immunoblot Analysis for Identifying Rabbits Infected with Trichophyton mentagrophytes

The aim of this study was to investigate the diagnostic potential of immunoblot analysis for detecting rabbits naturally infected with Trichophyton mentagrophytes. A protein extract of the dermatophyte T. mentagrophytes was prepared from a fungal mat grown in liquid medium, where the production of keratinases was stimulated by keratin as a nitrogen source. The proteins derived from disrupted T. mentagrophytes were separated by SDS-PAGE and visualized by Western blotting. Twenty-one rabbits infected with T. mentagrophytes and 10 non-dermatophyte exposed rabbits were studied. Eight dominant bands were identified in immunoblots from dermatophyte-infected rabbits, with apparent molecular weights ranging from 20 to 75 kDa. The diagnostic potential of the bands for identifying infected rabbits was evaluated by determining sensitivity, specificity, positive and negative predictive values, and overall accuracy. Bands at 20, 30 and 33 kDa were observed with 100 % specificity and 76.19 % sensitivity and can thus be considered highly accurate in correct identification of the infected animals. Accuracies from 54 to 62 % were found for the other dominant bands. Bands at 42, 45 and 75 kDa with 85.71 to 100 % sensitivity appeared not to be specific for dermatophyte infection. Although the specificity of bands at 26 and 28 kDa was found to be 100 %, their accuracy was only moderate, due to the low level of sensitivity, 9.52 and 23.81 %, respectively

Prosudba učinka alergen specifične imunoterapije u atopičnih pasa na osnovi CADESI-03 sustava bodovanja: kliničko istraživanje kontrolirano metilprednizolonom.

Allergen-specific immunotherapy (ASIT) is a method of specific treatment of atopic dermatitis (AD) which has been used for years in human and veterinary medicine. It is empirically known to be effective in dogs with AD, however, its true effectiveness still seems controversial due to insufficient evidence derived from controlled studies. The purpose of this study was to compare the results of an 8 month ASIT with 2-month symptomatic methylprednisolone treatment in dogs with AD. The third version of Canine Atopic Dermatitis Extent and Severity Index (CADESI-03) was used for the first time for objective evaluation of ASIT efficacy in dogs. 11 atopic dogs, older than 6 months, of various breeds and both sexes, were included in the study. Each dog was initially treated with methylprednisolone over a period of 2 months. After a washout period of 1 month, conventional ASIT with subcutaneous allergen injections was initiated and continued for the next 8 months. CADESI 03 scores were evaluated at regular monthly control visits. In both groups, ≥50% reduction of CADESI-03 score from baseline was recorded in 4/11 dogs (36,4%). A significant effect of ASIT was recorded after 6 months of therapy (P = 0.032). Thus, at least a period of 6 months is recommended before assessing treatment efficacy of ASIT. Methylprednisolone proved to be more efficient in controlling pruritus than ASIT. No remarkable changes in blood biochemical parameters were observed in either group. The results of our study suggest that the effect of 8-month ASIT in dogs with AD is comparable to that of 2-month therapy with methylprednisolone. The obvious safety of long-term ASIT additionally supports the more frequent use of this specific therapy in the treatment of canine AD.Alergen specifična imunoterapija (ASIT) metoda je specifičnoga liječenja atopijskog dermatititsa (AD) koja se godinama primjenjuje u humanoj i veterinarskoj medicini. Iskustveno je poznato da je učinkovita u pasa s AD, ali je njezina učinkovitost još uvijek prijeporna, jer nema dovoljno dokaza proizašlih iz kontroliranih istraživanja. Svrha ovoga istraživanja bila je usporediti rezultate 8 mjesečne ASIT s rezultatima dvomjesečnoga simptomatskoga liječenja AD pasa metilprednizolonom. Treća verzija određivanja stupnja i indeksa jačine atopijskoga dermatitisa u psa (engl. Canine Atopic Dermatitis Extent and Severity Index (CADESI-03)) prviput je bila rabljena za objektivnu prosudbu učinkovitosti ASIT u pasa. Jedanaest atopičnih pasa, starijih od šest mjeseci, različitih pasmina i različita spola bilo je uključeno u ovo istraživanje. Svaki je pas početno bio liječen prednizolonom tijekom dva mjeseca. Nakon jednomjesečne stanke, započeta je uobičajena ASIT sa supkutanom primjenom alergena u trajanju od sljedećih osam mjeseci. CADESI 03 bodovanje bilo je vrednovano prilikom redovitih mjesečnih kontrolnih pregleda. U obje skupine ustanovljeno je smanjenje CADESI-03 bodova ≥50% od bazičnoga u 4 od 11 pasa (36,4%). Značajni učinak ASIT-a bio je ustanovljen nakon šest mjeseci liječenja (P = 0,032). Stoga se procjena učinkovitosti ASIT-a može dati tek nakon liječenja od najmanje šest mjeseci. Metilprednizolon je bio učinkovitiji za suzbijanje svrbeža od ASIT-a. Nije bila ustanovljena značajna razlika u biokemijskim pokazateljima krvi među promatranim skupinama. Rezultati ovoga istraživanja pokazuju da je učinak 8-mjesečne alergen specifične imunoterapije u pasa s atopijskim dermatitisom jednak onome koddvomjesečnoga liječenja prednizolonom. Očita sigurnost dugotrajne ASIT dodatna je potpora sve češćoj upotrebi toga specifičnoga liječenja atopijskoga dermatitisa pasa

Učinak dijalize na peroksidaciju lipida i antioksidacijski status nerastove sjemene plazme pohranjene u tekućem stanju.

The aim of this study was to assess the effect of dialysis on the lipid peroxidation and antioxidant status in liquid stored boar semen. Seventeen ejaculates obtained from eight boars were divided into two aliquots. One aliquot was dialyzed; the other was used as a control. Semen was then stored at 16-17 °C for three days. Sperm quality parameters, capacitation status, thiobarbituric acid reactive substances (TBARS), superoxide dismutase activity (SOD) and total antioxidant capacity (TAC) of dialyzed and non-dialyzed semen were measured. The effect of lipid peroxidation and antioxidant status on semen quality was evaluated. Removal of low molecular weight components (LMWC) from seminal plasma by dialysis resulted in a signifi cantly higher percentage of morphologically normal spermatozoa (P = 0.002). Dialysis moderated the increasing level of acrosomal reacted spermatozoa, showing a significantly lower level of acrosomal reacted spermatozoa in dialyzed semen samples according to each day of storage (P<0.05). SOD increased with storage time in both groups, whereas control samples showed significantly higher levels of SOD than dialyzed ones (P<0.05). Dialyses yielded a significant decrease in TAC and an increase in the level of TBARS (P<0.05). Our results show that elevated levels of SOD in dialyzed semen are not able to counterbalance decreased levels of TAC and prevent oxidative injures to the spermatozoal sperm membrane.Cilj istraživanja bio je procijeniti učinak dijalize na peroksidaciju lipida i antioksidacijski status nerastova sjemena pohranjena u tekućem stanju. Sedamnaest ejakulata od osam nerastova bilo je podijeljeno u dvije skupine. Jedna skupina bila je podvrgnuta dijalizi, a druga je bila kontrolna. Sjeme je nakon toga bilo pohranjeno na 16 do 17 °C tijekom tri dana. Izmjereni su pokazatelji kvalitete sjemena: kapacitiranje spermija, reaktivne tvari tiobarbiturne kiseline, aktivnost superoksidne dismutaze i ukupna antioksidacijska sposobnost dijaliziranog i nedijaliziranog sjemena. Procijenjen je učinak peroksidacije lipida i antioksidacijski status na kvalitetu sjemena. Uklanjanje sastojaka male molekulske mase dijalizom iz sjemene plazme dovelo je do značajno povećanog postotka morfološki normalnih spermija (P = 0,002). Dijaliza je utjecala na razinu akrosomski reaktivnih spermija pokazujući njihovu značajno manju razinu reaktivnosti u dijaliziranim uzorcima po svakom danu pohrane (P<0,05). Aktivnost superoksidne dismutaze povećavala se s vremenom pohrane u obje skupine, ali su kontrolni uzorci pokazivali značajno veću aktivnost nego dijalizirani (P<0,05). Dijaliza je značajno smanjila antioksidacijsku sposobnost, a povećala reaktivne tvari tiobarbiturne kiseline (P<0,05). Rezultati pokazuju da povećane razine aktivnosti superoksidne dismutaze u dijaliziranom sjemenu ne mogu uravnotežiti smanjenu razinu antioksidacijske sposobnosti i spriječiti oksidativna oštećenja membrane spermija

Diagnostic Value of ELISA Tests for the Detection of Specific Antibodies in Cats and Rabbits with Dermatophytosis

Two indirect ELISA tests developed for the detection of specific IgG in cats and rabbits, infected with M. canis and T. mentagrophytes, respectively, were evaluated and compared. The levels of specific antibodies were determined in sera of 20 cats and 25 rabbits naturally infected with M. canis and T. mentagrophytes, respectively. Infection was confirmed by the results of fungal culture. Blood samples from 12 cats and 17 rabbits, previously unexposed to dermatophytes, served as negative controls. A significant increase in the level of specific antibodies in groups of infected animals was demonstrated. Sensitivity, specificity and predictive values of a positive and a negative test were determined to evaluate the diagnostic potential. ELISA for the detection of specific antibodies in cats infected with M. canis (ELISA-cats test) exhibited 75.0 % of sensitivity at 91.7 % of specificity, whereas the test for the detection of specific antibodies in rabbits, infected with T. mentagrophytes (ELISA-rabbits test) is highly sensitive (96.0 %) and highly specific (94.1 %), confirming its encouraging diagnostic potential. The cross-reactivity of fungal antigens was tested by performing the assays with antigens M. canis, T. mentagrophytes, M. pachydermatis and A. fumigatus. There were no significant indications of cross-reactions in the test T. mentagrophytes-rabbits, whereas strong cross-reaction between dermatophyte antigens was observed in the test M. canis-cats