Pitome vretice (lat. Mustela putorius furo) su mali sisavci koji pripadaju u porodicu kuna, te su uz pse i mačke jedna od tri pripitomljene vrste mesojeda. Prvotno su korištene za lov, a kasnije kao laboratorijski modeli. Njihov uzgoj kao kućnih ljubimaca započinje u ranim 80-tim godinama prošlog stoljeća, a popularnost im je u velikom porastu zadnjih četrdeset godina. U Republici Hrvatskoj pitome vretice postaju sve popularniji kućni ljubimci te im broj iz godine u godinu značajno raste. Endokrinološke bolesti smatraju se jednom od najčešćih skupina bolesti, a pojava endokrinoloških novotvorina kod pitomih vretica mnogo je veća nego u drugih vrsta domaćih životinja, među kojima su najčešće novotvorine gušterače i nadbubrežnih žlijezda. Glavnim uzrokom pojave adrenokortikalne bolesti (AKB) smatra se gonadektomija, koja ima za posljedicu izostanak supresije hipotalamusa i hipofize putem mehanizma negativne povratne sprege pri čemu dolazi do kontinuiranog lučenja gonadotropin oslobađajućeg hormona (GnRH) iz hipotalamusa koji stimulira hipofizu na lučenje luteinizirajućeg hormona (LH). Mogući utjecaj na pojavu AKB-a imaju genetski čimbenici, melatonin, fotoperiod i onkogeni putevi. Dijagnostika AKB-a uključuje karakterističnu kliničku sliku, mjerenje porasta koncentracije prekursora spolnih hormona u serumu, ultrazvučnu pretragu abdomena i patohistološku analizu uzoraka nadbubrežnih žlijezda. Budući da je AKB kroničnog karaktera, a da su istodobno prisutne bolesti (komorbiditeti) vrlo česte, bilo ih je potrebno uključiti u istraživanje. Ciljevi ovog istraživanja bili su utvrditi pojavnost AKB-a u pitomih vretica u Republici Hrvatskoj, utvrditi kliničke manifestacije AKB-a, utvrditi prisutnost komorbiditeta i njihov značaj te usporediti kliničke, laboratorijske i ultrazvučne nalaze s ciljem da se utvrdi prognostički značaj.
U istraživanje je bilo uključeno 70 pitomih vretica zaprimljenih na Kliniku za unutarnje bolesti Veterinarskog fakulteta Sveučilišta u Zagrebu u razdoblju od 20. travnja 2012. do 4. travnja 2018. godine. Ulazni kriteriji za istraživanje bili su slijedeći: da su sve jedinke gonadektomirane, da im je postavljena dijagnoza AKB-a na temelju prisutnosti simptoma i povišene koncentracije prekursora spolnih hormona (estradiol i/ili androstenedion), da je obavljena ultrazvučna pretraga abdomena, te da su prikupljeni uzorci krvi za hematološke i biokemijske (Suprelorin®, Virbac, Francuska) koji sadrži 4,7 mg deslorelin acetata. U istraživanoj populaciji je bila podjednaka zastupljenost oba spola. Pacijenti su pri postavljanju dijagnoze bili u dobi 2-8 godina, a najveći broj pacijenata je bio u dobi od 4 godine. Istraživana populacija podijeljena je u dvije dobne skupine, odrasle (mlađe od navršenih 5 godina) i stare pitome vretice (starije od 5 godina).
Najčešći simptom AKB-a je bila alopecija (80%), a zatim su po učestalosti slijedili promijenjeno ponašanje (21,4%), pruritus (18,6%), oteknuće stidnice (14,7%), strangurija (11,1%), urofagija (5,7%) i prisutan spolni nagon (4,3%). Hematološki i biokemijski nalazi pokazali su se korisnim u dijagnostici komorbiditeta i komplikacija AKB-a. Ultrazvučna pretraga abdomena je bila korisna za potvrdu definitivne dijagnoze AKB-a i za utvrđivanje komorbiditeta u trbušnoj šupljini. Komorbiditet je zabilježen u 53, a višestruki komorbiditeti u 8 pacijenata. Početni komorbiditet je zabilježen u 32, a naknadni u 29 pacijenata. Početni i naknadni komorbiditet bio je prisutan u 8 pacijenata. Na vjerojatnost preživljenja nije utjecao spol, ali je postojala značajnost u preživljenju s obzirom na dob i prisutnost komorbiditeta (početni i/ili naknadni komorbiditet). Medijan preživljenja u skupini odraslih pitomih vretica iznosio je 1183 dana (95% CI 984-1411), a u skupini starih pitomih vretica 408 dana (95% CI 256-655). Medijan preživljenja skupine pacijenata bez početnog komorbiditeta iznosio je 1120 dana (95% CI 779-1273), a u onih s početnim komorbiditetom 509 dana (95% CI 267-762). Medijan preživljenja u skupini bez naknadnog komorbiditeta iznosio je 990 dana (95% CI 581-1278), a u onih s naknadnim komorbiditetom 600 dana (95%CI 199-984). Smrt posljedično komorbiditetima bila je više zastupljena (71%) nego smrt posljedično AKB-u, dok je kod pojedinih pacijenata s komorbiditetima uzrok smrti ipak bio AKB. Analizom Kaplan-Meier krivulje preživljenja izrađen je nomogram vjerojatnosti preživljenja pitomih vretica u odabranim vremenskim intervalima, s obzirom na dob i prisutnost početnog i naknadnog komorbiditeta koji će se rabiti u prognozi ishoda bolesti za pojedinačne pacijente.
|Abstract (english)|| |
The domestic ferret (lat. Mustela putorius furo) belongs to the Mustelida family and is one of the three domesticated carnivore species along with dogs and cats. Ferrets have most probably been domesticated in southern Europe more than 2000 years ago. Initially they were used for hunting and later as laboratory models. In biomedical researches ferrets have been used since the beginning of the 20th century, mostly due to the similarities with humans and susceptibility to many human pathogens and diseases.
Nowadays ferrets are popular as companion animals worldwide, and their popularity is remarkably increasing in the last decades. In Croatia, ferrets are also popular pets since 1990’s with intense increase in their number during the last two decades. The number of ferrets presenting as patients has also doubled, posing new challenges to small animal clinicians due to their unique species-specific pathology which imposes species-oriented approach in diagnostic and treatment techniques. Endocrinological diseases are frequently seen in ferrets, notably more frequent than in other domestic animal species. Endocrinopathies are the most common non-infectious diseases affecting middle-aged and older ferrets with pancreatic and adrenocortical neoplasms being predominant. Diet, gonadectomy and genetics may predispose ferrets to an increased incidence of these endocrinopathies. It is also suggested that not only genetic factors but also melatonin, duration of photoperiod and oncogenic pathways play an underlying role in the development of adrenocortical disease (ACD).
The main cause of the ACD is gonadectomy which disrupts negative feedback mechanism on hypothalamus and pituitary gland and results in secretion of gonadotropin-releasing hormone (GnRH) which ensues and subsequently stimulates pituitary gland to release luteinizing hormone (LH). The disease itself is characterized by unilateral or bilateral neoplasia or hyperplasia of adrenocortical tissue that expresses the LH receptors. These receptors are found in clear cells of the zona glomerulosa and zona fasciculata in the adrenal glands of healthy ferrets and those with the ACD. However, it seems that the LH receptors are functional only in the adrenal glands of affected ferrets because plasma concentrations of adrenal androgens do not increase after intravenous injection of gonadotropin releasing hormone (GnRH) agonist in healthy ferrets. The adrenal cortices of affected ferrets produce a variety of sex hormones such as estradiol, androstenedione and 17 α-hydroxyprogesterone. The clinical features of the ACD include symmetrical alopecia, pruritus, vulvar swelling and seromucous discharge in neutered females, prostatomegaly and cysts in males that can lead to stranguria, dysuria, pollakisuria and tenesmus during defecation. Trauma and preputial dermatitis can also be present. Behavioural changes encompass recurrence of sexual behaviour and aggression after neutering, mainly in males. A diagnosis of the ACD is usually based on combination of typical clinical signs, diagnostic imaging, complete blood count and blood chemistry analysis and is confirmed on the basis of elevations in plasma estradiol, androstenedione and/or 17- hydroxyprogesterone. Results of complete blood count and biochemical parameters are usually normal and unremarkable. An abdominal ultrasonography is the method of choice for demonstrating changes in the adrenal gland size and echogenicity.
There are two treatment options for the ACD in ferrets, surgical and medical, both aimed towards reduction in sex steroide concentrations. The aim of the surgery is to remove or debulk the tumour (adrenalectomy). On the other hand, even though medical treatments do not affect the tumour and thus are not curative, they prevent surgical risks and cause longer remission. The most effective drugs at this moment are the GnRH-analogue depot Suprelorine® and the GnRH-agonist depot leuprolide acetate Lupron Depot®. The decision whether to choose medical or surgical treatment is individual and based on various factors (which gland is affected, severity of clinical signs, patient’s age, concurrent diseases, surgeon's experience and owner's finances). The goal of the treatment is to provide longest possible life span with a good life quality. As the ACD is a chronic disease, with proper and early treatment, affected ferrets can survive for a long time and therefore can acquire many comorbidities (which can be life threatening).
The aim of this study was to determine the incidence and clinical manifestations of the ACD in ferrets in Croatia, as well as the presence of comorbidities with their significance, and to compare the clinical, laboratory and ultrasonographic results in order to determine their prognostic significance.
The study included 70 ferrets admitted to the Clinic for internal medicine, Faculty of veterinary medicine of the University of Zagreb, during the period between April 20th 2012 and April 4th 2018. Inclusion criteria were the following:
1) previously performed gonadectomy,
2) confirmed diagnosis of ACD based on clinical symptoms and increased concentration of at least one of the sex hormone precursors (estradiol and/or androstenedion),
3) abdominal ultrasound performed and
4) blood samples for haematology and biochemistry obtained.
After establishing the diagnosis, all ferrets were subcutaneously implanted with Suprelorin® (Virbac, France) containing 4.7 mg of deslorelin acetate once a year. For the purposes of this study, two age groups were defined: adult ferrets (younger than 5 years of age) and old ferrets (older than 5 years at the time of diagnosis of the ACD). In order to monitor survival and outcome it was necessary to include comorbidities in this study. Hence, potentially life-threatening diseases that frequently occur in ferrets (insulinoma, lymphoma, heart disease, pneumonia, renal disease, liver disease) were investigated. The term “preliminary comorbidity” was considered as the comorbidity that was present at the time of the ACD diagnosis while the term “additional comorbidity” was considered as comorbidity that occurred after ( 14 days) the diagnosis of the ACD. Survival and the causes of death were investigated with possible outcomes (for each individual patient) as death due to the ACD or death due to comorbidity. Censored patients were those whose death was not caused by either the ACD or comorbidity, or the ones that were alive at the end of the study, lost to follow-up, or died due to other causes (e.g. trauma).
There was an equal representation of both genders aging from 2 to 8 years with highest incidence of the ACD occurring at the age of 4. The most common clinical symptom of the ACD was alopecia (80%) followed by changed behaviour (21,4%), pruritus (18,6%), vulvar swelling (14,7%), stranguria (11,1%), urophagia (5,7%) and sex instinct drive (4,3%). There were 75,7% of patients with comorbidity noted and 11,4% with multiple comorbidities. Preliminary comorbidity existed in 45,7% of the patients while additional comorbidity was developed in 41,4% of the rest. Both preliminary and additional comorbidities were present in 11,4% of patients. The results showed that the probability of survival was not influenced by gender, but that there was significance in survival with respect to the age as well with the presence of comorbidity (preliminary and/or additional). The median survival in the group of adult ferrets was 1183 days (95% CI 984-1411) and in the group of old ferrets 408 days (95% CI 256-655). The median survival in the group of patients without preliminary comorbidity was 1120 days (95% CI 779-1273) and in those with preliminary comorbidity it was 509 days (95% CI 267-762). The median survival in ferrets without additional comorbidity was 990 days (95% CI 581-1278) and in those with additional comorbidity it was 600 days (95% CI 199-984). The results showed that death due to comorbidity (71%) was prevalent in comparison with death due to the ACD. A nomogram of the survival probability in ferrets affected with the ACD at selected time intervals, given the age and the presence of preliminary and additional comorbidity, was created by Kaplan-Meier survival curve analysis in order to be used in the prognosis of the ACD outcome for individual patient. Statistical and exploratory data analyses were performed using R v4.0.4 (R: A language and environment for statistical computing, R Core Team (2021.), R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/). Exploratory analyses included standard measures of the central tendency and dispersion, and normality of the data were evaluated using Shapiro-Wilk's test. Normal data were analysed using ANOVA for comparisons of more than 2 groups and using student's t test for 2 groups (including F test of homogenity of variance for application of Welch's correction of degrees of freedom). Non-normally distributed data were analysed using Kruskal-Wallis test for more than 2 groups, and Mann-Whitney test for 2 groups. In case of multiple comparisons post hoc tests included pairwise t test and Dunn's test for normally and non-normally distributed data, respectively. Categorical data were explored using counts and proportions, and analysed using Chi square and Fisher's test as indicated by cell sizes of contingency tables. Analysis of survival data were performed using log-rank tests of Kaplan-Meier survival curves and Cox proportional hazards regression models. Models were built using the forward step procedure and selected based on ANOVA tests. Cox regression model diagnostics included evaluation of coefficients as a function of time using scaled Schoenfeld residuals for assumption of constant proportional hazards, dfbeta residuals for influential observations, and martingale residuals for functional form of covariates. Demographic variables not observed significant in univariate tests were evaluated as strata in multivariate analyses. The analysis of cause of death was performed using logistic regression with modelling performed as for Cox regression models. The diagnostics of logistic regression models included evaluation of residuals, influence and leverage points and model validation using 100 bootstrap replicates. Model's confusion matrix was analysed using receiver-operating characteristics for evaluation of diagnostic performance characteristics. For correlation analyses of haematological and biochemical data Spearman's correlation test was used. All tests used alpha error set at < 0,05.
Adrenocortical disease is frequent disease in ferrets in Croatia affecting middle-aged and old ferrets without sex predisposition. Alopecia is the most frequent clinical sign which can be present in either localized or generalized form. Haematological and biochemical parameters are helpful in investigation of comorbidities but not for the ACD diagnostics. Measurement of hormone precursors concentrations represents a golden standard for the ACD confirmation, with the increase, of at least one, being sufficient. Abdominal ultrasound is useful in diagnostics of both, the ACD and comorbidities. The most common comorbidity was insulinoma with incidence of 38,6%. Patient’s age, preliminary and additional comorbidity were significant for survival time of ferrets affected with the ACD. In conclusion, elderly patients with both preliminary and additional comorbidities had shortest survival times. A nomogram adapted to ACD shows good results in predicting the probability of survival in ferrets suffering from ACD.