| Abstract | Pseći herpesvirus-1 (CaHV-1) enzootski je prisutan u pasa širom svijeta i često se navodi kao značajan uzrok uginuća štenadi, pobačaja i drugih reproduktivnih poremetnji. Do danas ne postoji opće prihvaćena metoda koja bi služila kao zlatni standard u dijagnostici infekcije CaHV-1. Također, do danas u Republici Hrvatskoj nije provedeno sustavno istraživanje seroprevalencije i epizootiologije CaHV-1 niti su rađene poveznice s reproduktivnim poremetnjama u kuja.
Cilj ovog rada bio je pronaći optimalnu metodu serološke dijagnostike infekcije pasa s CaHV-1, njezin značaj i proširenost na području Republike Hrvatske. Također, cilj rada bio je dobiti uvid u epizootiološke značajke ove infekcije. Podaci prikupljeni za potrebe ovog istraživanja iskorišteni su za ispitivanje utjecaja izloženosti kuja CaHV-1 i povezanosti s poremetnjama u reprodukciji, tijekom i ishodom gravidnosti i okota. Kao dodatni dio ovog istraživanja, pokušalo se dati odgovor o učinkovitosti imunoprofilakse u suzbijanju infekcije CaHV-1.
Seroprevalencija CaHV-1 u uzgajivačnicama, određena virus neutralizacijskim testom, iznosila je 32,02% i 30% određena imunoenzimnim testom. Statistička analiza pokazala je da se CaHV-1 u uzgajivačnicama širi i horizontalnim prijenosom u kuja i unosom uzročnika u uzgoj nakon parenja muških životinja. Kritični čimbenici u epizootiologiji infekcije CaHV-1 su: veličina uzgoja, sudjelovanje na izložbama, lov i provođenje dnevne dezinfekcije. Međutim, serološki status kuje nema značajan utjecaj na pojavu reproduktivnih problema, na način okota kuje, niti na broj štenadi prilikom okota. Serološki status kuje ne utječe na točnost predviđanja dana okota (putem pada vrijednosti serumskog progesterona, rektalne temparature niti na fetometrijska mjerenja). Također, nije utvrđen stadij spolnog ciklusa u kojem je statistički značajno veći broj seropozitivnih kuja. Od 40 cijepljenih kuja, 52,50% životinja nije serokonvertiralo, na što značajno utječe da li je kuja cijepljena u ambulanti ili izvan nje.
Visoka seroprevalencija CaHV-1 u Republici Hrvatskoj je očekivana. Znanstveni doprinos ovog rada je u određivanju optimalnog postupka izvođenja VNT koji ima veću osjetljivost od postupaka koji su ranije opisivani u literaturi. Analizom čimbenika rizika vidljivo je da za suzbijanje infekcije CaHV-1 je potrebno držanje kuja u uzgajivačnicama u manjim skupinama i provođenje mjera opće profilakse koje spriječavaju horizontalno širenje infekcije. Za rasplodne mužjake najbitnija profilaktička mjera je spriječavanje zaražavanja
prilikom parenja. Rezultati su pokazali da imunoprofilaksa nije uvijek učinkovita i da se edukacijom uzgajivača osobito treba utjecati na suzbijanje prakse da se životinje cijepe od strane nestručnih osoba.
pseći herpesvirus-1, serološka i molekularna dijagnostika, čimbenici rizika, reproduktivne poremetnje |
| Abstract (english) | Canid alphaherpesvirus-1 (CaHV-1) is enzootic pathogen of the dogs often related to abortions, neonatal mortality and other reproductive disorders. Unfortunately, until today there is no generally accepted method that would serve as a golden standard in diagnostics of CaHV-1 which makes control of the disease very difficult. Also, to date there were no researches on CaHV-1 seroprevalence and epizootiology in Republic of Croatia and the influence on the reproductive disorders has not been investigated.
The aim of this study was to determine optimal serological method which could give the most realible results in diagnostics of CaHV-1. Also, the aim was to detemine seroprevalence in Republic of Croatia and risk factors which could influence a seroprevalence, CaHV-1 antibody titre in breeding kennels and excretion of the virus in latently infected animals. Statistical analysis and modelling was used to give insights in possible influence of CaHV-1 infection in dams on reproductive disorders. Comparison of seropositive and seronegative dams in the last 48 hours before whelping was aimed to find the influence of seropositivity on the physiology and pathology of whelping and on the number of the live puppies.
METHODS
In the period from 2014 to 2017 a total of 136 breeding bitches and 67 stud dogs, with no history of vaccination, were randomly sampled from 26 different breeding kennels in Croatia. From each animal multiple sera samples, nasal, vaginal and preputial swabs were collected. For the purpose of statistical analysis data regarding kennel size, attendance at dog shows and hunt trials, daily disinfection protocol, gender, age, breed, number of mattings and a stage of estrus cycle in dams were collected. In bitches, additional sera samples were collected in different stages of oestrus cycle and in 13 dams from one breeding kennel with reproductive problems late stage of the pregnacy was followed using ultasound and X-rays. Also, serum progesterone and rectal temperature values was mesured in last 48 hours before whelping. A type of whelping and number of alive and stillborn puppies was followed. All data were compared with a serological status of the dam.
Especially for this trial, three methods of virus neutralisation assay (VNT) were introduced and 135 sera samples were compared. Also, 89 sera samples were tested with ELISA method, using commercially available kit.
In addition to original study plan sera samples of 40 bitches vaccinated against CaHV-1 were collected in order to examine efficiency of the vaccination protocol. Data regarding vaccination date, number of vaccinations and veterinary organisation where vaccinations took place were collected and analysed.
RESULTS
Overall seroprevalence of CaHV-1 in 203 breeding dogs in Croatia using VNT was 32,02% and usine ELISA was 30%. Of the three methods of VNT (using native serum, heat inactivated serum and heat inactivated serum samples with addition of complement) the highest sensitivity was achieved using the native serum samples. Results of the VNT were compared with ELISA with a low correlation between tests. Excretion and shedding of the virus using molecular methods was not proven.
Statistical analysis showed that CaHV-1 in breeding kennels spreads and establishes enzootic cycle in two ways. In dams CaHV-1 is introduced trough infections at dog shows and later is spread horizontally in kennel. In the breeding males infection is the most often acquired during mating. In both genders, hunt was very important activity for seroprevalence and a maintance of the present antibody titre. Critical risk factors in epizootiology of the CaHV-1 infection in the breeding kennels were: a kennel size, attendance at dog shows, attendance at hunt and kennel disinfection protocols.
There was no significant influence of the oestrus cycle on the serological status of the dams. Pregnancy and lactation did not affect seroprevalence or the level of CaHV-1 antibody titres. Also, correlation of the serological status of the dam and reproductive problems, type of whelping (cesarean section or a natural whelping) or number of live, dead and on the total number of born puppies and prediction of the whelping date (via progesterone, rectal temperature and fetometry measurements) could not be established.
As additional part of this study, seroconversion in 40 vaccinated dams was followed. Of all vaccinated dams, a significant number (52,50%) of the dams did not seroconvert.
CONCLUSIONS
A high seroprevalence of CaHV-1 in Republic of Croatia was expected and it was in a line with a published results in other countries. Important scientific impact of this study is optimisation of the VNT procedure which showed that using native seras has stronger sensitivity than other methods described in literature and it is a method of choice in serological diagnostics of CaHV-1.
In analysis of the risk factors it is shown that two aproaches are needed in prophylaxis of CaHV-1 infection. In dams is very important to make a smaller groups in kennels and to use daily cleaning which can stop spreding of the disease. For the stud dogs the most important prophylactic measure is prevent infection during mating. Unfortuntaly, datas from this work has shown that specific immunoprophylactic measures are not always efective. Also, education of the dog breeders is necessary to point out that veterinary pactitioners should do the vaccination. |
