FANDING OF PARASITIC NEMATODES OF FISHES PRESENT IN THE MARKET

When placing the fish and fish products on the trade it is necessary to pay attention to the presence of zoonotic parasites that can lead to infection of people especially if the fish is consumed raw or undercooked. Epidemiologically the most important are helminths from the group of Nematoda. The most important are Anisaxis spp. and Eustrongylides excisus. Anisakiasis is a serious zoonotic disease with a dramatic increase in prevalence throughout the world in the last two decades. The larvae are found in marine fish species most frequently in sardines, herring and mackerel. Eustrongylidosis is a disease that occurs primarily in freshwater fish species: catfish, zander and pike, a cause is a parasite of the genus Eustrongylides of which the most important species is Eustrongylides excisus. In this paper, the nematodes that occur in fish that are present in the market in the Republic of Serbia are present. Investigations of Anisaxis spp. were carried out at the Institute of Veterinary Medicine Novi Sad in the period of 2000-2013. Total of 2414 samples of imported marine fish was inspected. In 25 (1.29 % ) samples of herrings (Clupea harengus ) and mackerels ( Scomber scombrus ) was identified Anisakis spp. Investigations of Eustrongylides spp . were carried out in the period 2010-2013 at the Danube- Tisa -Danube Canal. Samples were collected from zander (Sander lucioperca) and European catfish (Siluris gla nis) in which the presence of nematodes was found in the abdominal cavity, muscles, the lumen of the stomach and gastric wall where the parasites were encapsulated. Individuals of zander were examined during the 2013 at the Veterinary Institute Novi Sad, where the presence of larvae Eustrongylides spp.u muscle was detected. In order to avoid infecting people with parasites of fish it is necessary to carry out continuous control and monitoring. Fresh


INTRODUCTION
Zoonotic nematodes infect freshwater and marine fish species.Pathology normally occurs within the intestines but can affect all organs.Nematodes can infect fish as adults but larval stages of nematodes infecting piscivorous birds, mammals or reptiles, or less frequently predatory fish, can also infect fish species (Roberts and Janovy, 2000).Some nematodes are zoonotic.Аmong these parasite Anisakis spp.has the highest medical importance because of the severe allergic reactions and gastrointestinal symptoms it causes in humans after eating or handling infected fish or crustaceans (Lima dos Santos and Howgate, 2011).These symptoms are especially prevalent in countries where it is common to eat raw or undercooked fish.Anisakiasis a serious zoonotic disease with a dramatic increase in prevalence throughout the world in the last two decades.People who are usually accidental hosts infected when ingest the third larval stage of the parasite that can be found in the internal organs and muscles of a large number of marine and anadromous fish (sardines, herring, salmon, tuna, mackerel, etc.) (Bullini, 1997;Oliva, 1999).Definitive host of Anisaxis species are marine mammals commonly dolphins and whales in which intestines are adult forms of the parasite.The first intermediate hosts are marine crustaceans and second intermediate hosts are cephalopods and fishes.Anisakis simplex is present in Atlantic, Pacific, Mediterranean Sea, Norwegian Sea and Barents Sea.The highest prevalence of anisakiasis is found in nort Asia and western Europe (Netherlads, Germany, France and Spain) (Chai et al., 2005).In Serbia Anisakis can be present in imported marin fish.
The most significant freshwater fish zoonotic nemotods which occurs in Serbia are Eustrongylides species (Ljubojević et al., 2012: Novakov et al., 2013).Thay have complex life cycles involving a definitive host and two intermediate hosts.Definitive hosts include aquatic birds mostly from order Ciconiiformes family Ardeidae, Anseriformes, Gaviiformes and Pelecaniformes (Spalding

RESULTS AND DISCUSSION
Anisakis spp was first isolated in the laboratory of Veterinary Institute of Novi Sad in 2003.On a total of 2414 samples of imported marine fishes, in 25 (1.29%) was found Anisakis spp.Presence of Anisakis spp. in samples of imported fish from 2000-2013 are shown in table 1 and chart 1. Coiled nematodes were present in herring (Clupea harengus) and mackerel (Scomber scombrus) (Fig. 1).Common to all positive samples is the presence of the viscera, because fishes were not eviscerated after fishing.Number of parasites ranged from 2-3 to 52 nematodes per fish.In two samples (0.08%) the presence of larvae in fish muscles was performed by the method of artificial digestion.(Spalding et al., 1993).Second intermediate hosts are planktivorous and benthivorous fishes that could pass the infection on to fishes (paratenic hosts) and finally on to fish-eating birds (Moravec, 1994).Such exposure is usually common in larger fish species, like channel catfish -Ictalurus punctatus or pike-perch -Sander lucioperca, which, as predators, become infected with Eustrongylides spp.nematodes.In fish, these parasites are conspicuous as long, red, coiled individuals located in the body cavity or embedded in the muscle (Mitchum 1995, Overstreet 2003).
In humans who have consumed raw or undercooked fish, Eustrongylides spp.have produced gastritis and intestinal perforation (Deardorff and Overstreet 1991; Cole 1999).Guerin et al., (1982) were the first to report a natural (accidental) human infection with Eustrongylides spp.
The goal of this paper is to distinguish the presence of Anisakis spp.and Eustrongylides spp.nematodes in marine and freshwater fish species that are present in the market and inland waters of Serbia and to indicate the need for adequate preparation of fish meat.

MATERIAL AND METHODS
Investigations of Anisaxis spp.were carried out at the Institute of Veterinary Medicine Novi Sad in the period of 2000-2013.Total of 2414 samples of imported marine fish was inspected.Each fish was eviscerated and carefully examined.In same cases muscles of fish were checked by method of artificial digestion.
Diagnostics and investigations of Eustrongylides spp.larvae were conducted from 2010-2013.Fifty two fish samples of European catfish (Siluris glanis) weighing 250-450 g and twenty one samples of zander (Sander lucioperca) were collected from different locations on Danube-Tisa-Danube Canal.Fishes were parasitologically examined through cutting the body and the abdominal part, digestive tract and other ventral organs.Samplaes of zander was also examined at the Institute of Veterinary Medicine Novi Sad during 2013.Collected nematodes were were fixed in 70% ethanol for 24 h and cleared in lactic acid for morphological observation.Relative parametars were measured and identification was performed using Bauer (1987), Moravec (1994) and Anderson (2000) keys.
in stomach in 4 individuals of zander and 6 individuals of European catfish, what represented the prevalence of 14.26%, respectively 11.54%.During the 2013 at the Institute of veterinary medicine of Novi Sad larvae of Eustrongylides spp.were detected in the muscles of four samples of zander (Fig. 3).The number of parasites per fish ranged from a few up to the 256.This larva, was robust and pinkish red.Length of body was 27 -60.5 mm, maximum width 0.49 -0.58 mm.Presence of Eustrongylides spp.nematodes was revealed in the abdominal cavity (Figure 2), musculature , in the lumen of the stomach and encapsulated

CONCLUSION
Zoonotic nematodes can lead to human infection and should pay special attention on them.Fresh fish meat and traditional fish product should be subjected to a visual examination for the purpose of detecting visible nematodes and other parasites before being placed in the trade.The consumption forms and the preparation of the fish food should be modified in a way that hazards to human health due to zoonotic parasites could be avoided.Health education is a key factor in combating zoonotic infections.Fish parasites such as Anisakis spp.and Eustrongylides spp.are highly important because they are capable to infect humans (Mohammad et al., 2011).Murrell (2002) suggested several control measures for preventing parasitic infections originating from freshwater, such as environmental control of surface water, hygienic aquaculture, and the control or elimination of the first intermediate hosts.FDA (2001) indicated that the effective methods for killing parasites are freezing, heating, adequate combination of salt content and storage time or hot smoking.On the other hand, brining and cold smoking may reduce the parasite hazard in fish, but they do not eliminate or minimize it to an acceptable level (Murrell, 2002).While health education is a key factor in combating zoonotic infections, experience in various countries has shown that for successful implementation of control measures, it is necessary, as Hughes (1992) points out, to have formal and informal cooperation between medical and veterinary interests at all levels of government, and with the community.According to Okumura et al., (1999) and Chieffi et al., (1992), the recommendation to avoid consumption of raw or poorly cooked fish is still the best preventive procedure.

INTRODUCTION
Q-fever is an infectious disease which is the most common of all rickettsia caused diseases in the world and also in our country (2,4,8,10,12).This antropozoonozes is characterized with a wide spectrum of hosts and vectors.Primary role of domestic animals is as reservoirs for the infection of humans and therefore it is very important to precisely and in time Q-fever is detected in domestic animals (3,5,6,17).C. burnetii is one of the most resistant of all unsporalizing microorganisms.In tick feces, it can last for 586 days, in dried blood at room temperature it can last for 5 months, in dust 120 days in wool 12-16 months at 4-6 C, and in milk more than 30 days ( 13).
Sanitation of endemic foci of Q-fever is practically pretty close to impossible because of the high resistance of the causative agent of Q-fever, a small infectious dosage and various epidemiologies.Spreading of the disease via ticks as vectors is not significant, but inhalation of the causative cause together with the dust is.For development of an infection in domestic animals, a primary cycle in the nature is important.Positive reactors are found in sheep, cattle goats, swine, horses, poultry and cats (4,9,14,25) .From clinical point of view, Q fever is not a negligible issue in veterinary medicine.The miscarriages have been registered, primarily in the sheep, cattle and goats, but also other reproductive disturbances are present, mastitis, poorly viable offspring, etc. (19,20,22).The significance of some domestic animals in epidemiology of Q-fever is different in certain regions and it depends on their number, level of infection, size of the herd, way of breeding and zoo hygiene.The epidemic movement of the Q-fever disease is influenced by other factors such as air flow, rainfall, and density of population and geological characteristics of the terrain (17,24).
Alimentary infections are far less significant compared to the airborne infections (16).The cause of these infections is usually contaminated milk and milk products.Among all food, milk carries the biggest risk of infection.Milk pasteurization is a prophylactic measure for excluding every possible risk of infection for humans.
The problem of prevention from Q-fever is in the lack of specific protection measures and poor efficiency of general preventive measures.Habits of the mals, restriction of milk and wool, adequate hygiene, using the protective clothing, desinfection of equipement and vechicles, removing of placenta particles, removing of manure, quarantine after calving and lambing and not use common grazing.People proffesionally exposed to the risk from infection should be educated about the disease.

Situation in AP Vojvodina
Epizootiological study in our country shows that the prevalence in sheep is higher than the prevalence in cattle ( 11,17,21).Nomad farming has a special significance in the occurrences of epidemics.In Vojvodina, Q-fever is being maintained endemoepidemicaly (15,17).
Results after a study on the presence of Q-fever on cattle farms in Vojvodina show a significant presence (9,5%), while in certain farms the percentage of seroreactors is even over 30% (9,14,25).The highest percentage of seropositive findings was detected in cows which had miscarriages (l9, 4%), and the lowest was found in animals from endemic regions (4, 8%) (19,20).The results from serological analysis of workers at cattle farms with enzootics of Q fever show the presence of high level of risk from the infection with C.burnetii, although none of the seropositive workers did not report that he or she was ill from Q fever.Since Vojvodina is an endemic region for Q fever, risk level was analyzed from the infection among users of the raw milk in Vojvodina population (16).A significant difference was not found between the rate of infection in raw milk users (10,81%), compared to the rate of infection in the rest of the population (9,16%).There are literature data on epidemics of Q fever among cattle farm workers, where the infections were in mild form or without clinical symptoms of the disease (2,13,16).The significance of sheep in the epidemiology of Q fever in Vojvodina is analyzed based on a topographic distribution of registered cases of Q fever in humans, results of serological analysis of 1340 sheep blood samples and 1398 blood samples from the residents of villages.Measured with the percent of positive reactors, sheep from individual economies (sector) have a greater epidemiological significance.Seroprevalence in the sheep from individual sector (13,8 %) is significantly higher compared to the seroprevalence in the sheep from farms 9,9 % (X= 4,593).A statistically significant (X=94,287) higher percentage of seropositive animals was found in the regions where Q fever epidemics were registered (31,3 %), compared to the seroprevalence of sheep in other regions (8,3 % ).The significance of sheep in the epidemiology of Q fever is confirmed by a significantly higher (X=16,606) seroprevalence in village residents (20,3 %), who are professionally or in some other way in contact with sheep, compared to the residents without the con-program application, with remark that this period should be prolonged.As a key factor to the success, vaccination should be done not only for a short period of time.During the first year of vaccination there is a reduction of clinical symptoms (abortions, infertility), but vaccination period of 3-4 years is necessary in order to stop the shedding of bacteria.Vaccination program for cows and heifer is considered more efficient then program only for heifers.After application of vaccine C.burnetii phase I, heifers vaccinated before pregnancy have five times less probability of getting the infection then already pregnant cows (1,2,13,18).These results justify the application of the vaccination programs for animals in non infected herds and heifers before pregnancy.Besides, vaccination of cows should be done when the level of seroprevalence is low, meaning in herds of cows where the infection did not spread yet.In infected herds, vaccination should be done in all the animals or at least heifers.Vaccination of chronically infected cows prevents shedding of rickettsia through milk, constant or intermittent shedding.Vaccinated animals are considered not infected regardless to the specific antibody titer level.Vaccination does not aim only protection of the animals from infection, but also it eliminates shedding of Coxiella with milk (2,13).Efficiency of the application of all control measures including vaccination should be estimated with application of serologic test and PCR methods by systematic sampling of blood, milk, vaginal mucus and feces.Application of vaccination has given a new concept to the suppression of this zoonozes not only in the service of public health but also for creating regions free of Q fever in endemic regions (13).
After vaccination a problem of local reactions on the place of inoculation can appear, in sensitized animals and also in non sensitized ones as we have found in our research.In our study we did not detect rickettsia in milk samples from vaccinated cows 45 days after infection.Non vaccinated animals were continuously shedding rickettsia during the whole period of study.Today there are several vaccines at disposal for application in cows and sheep, such as bivalent vaccine C.burnetti and Ch.psittacci for sheep.Higher efficiency (300 times) was shown after vaccination with vaccines made of C.burnetii phase I, more virulent (has a complete LPS) then vaccine made of a virulent strains in phase II.Strains at disposal are usually Nine Mile strain, and rarely Henzerling strain for vaccine preparation.There are no unique programs for animal's vaccination; it is done related to the epidemiological situation in a certain region.Vaccination as a measure of prevention is used in Australia for sheep and humans exposed to the risk.In Russian federation vaccination is done in certain regions where Q fever appears endemically, where infection foci are present in the nature and in France and Slovakia, depending on the epidemiological not have a seasonal character any more.The disease has kept a characteristic demographic distribution with a highest specific incidence for productive population and males.More frequently the disease is found in males because of the higher rate of exposure, but also because of the difference in clinical manifestation of the disease.A study was done on 323 patients with acute infection and it was determined that asymptomatic infections were significantly more frequent in female patients (14,0%) then in male patients (4,3%) (15,17).In a study on the prevelance level for C. burnetii in population of AP Vojvodina in the middle 80es, the difference between the level of prevelance in different genders was not found (9,4% for males and 9,3% for females), while the average incidence for Q fever in males was 2,6 times higher (12,3/100.000)compared to the females (4,8/100.000)(15,,17).Even when the incidence is significantly reduced, the number of male patients (1,10/100.000) is 2 times higher compared to females (0,55/100.000)

CONTROL
General preventive measures give insufficient results in suppression and prevention of Q fever infection and specific prevention has no adequate solutions in the whole world.Great resistance of Coxiella in the environment and different epidemiology lead to almost impossible sanitation of endemic focus.Within the control program against Q fever the most frequently suggested measures are serological examination and vaccination of animals (2,13,23).One of the measures is removing of positive reactors from the flock and in other animals constant control is needed and separation of seropositive animals.Milk from seropositive cows must be pasteurized.Several authors have determined positive effects after vaccination in infected and non infected animals.
Q fever control in cattle is of a great significance, because of the presence of reproductive failures and because they are a significant source of infection and environment contamination ( 2,12,13,18,23).In practice, vaccination is recommended in infected herds and flocks, but a study on efficiency of different vaccination protocols has not been done, which is related to length of vaccination program, which categories of animals should be vaccinated and when (18).The latest research show good results on the level of single animal and on the level of flock or herd.Analyzed parameters were excretion of the causative agent, contamination of the environment and health status of the animals with a most important aim to determine measures for prevention and suppression of the infection.After application of vaccination a significant decrease of infection level was found during the first years of the vaccination -Regular cleaning an disinfection during lambing or calving -Rodent control and tick control -Reduce the dust -Use of quarantine when purchasing new animals -Determine the causes of abortion and still birth -Restrict entrance of people and other animals (including dogs and cats) whenever it is possible Pregnant women, immunocompromised persons and persons with cardiovascular problems (especially if there is a valve failure) should avoid close contact with the animals, particularly during the partial season.It is recommended to use pasteurized milk and use of pasteurized milk for milk products (cheese).There should be a restriction order to objects or space which is of high risk for Q fever in humans and entrance should be allowed only to vaccinated humans.
Immunization of humans with a high risk occupation is a primary preventive step against Q fever (13).There is a certain risk from serious local reactions to vaccine in humans which were exposed to the infection or were vaccinated.That is why before vaccination it is important to: -Check in anamnesis if the person had Q fever, or had clinical symptoms characteristic for Q fever or was vaccinated -Analyze blood serum for the presence of antibodies against C.burnetii or -Perform an intradermal test Every positive result after screening preludes vaccination.Vaccination induces a solid immunity in majority of vaccinated individuals.Good communication on local and regional level and also cooperation between human and veterinary medicine is necessary because of the significance of this disease.

EXPERIENCE FROM HOLLAND
Publicity was informed about the event in Holland when there was a great Q fever epidemic registered in humans (7,13).It all begun in 2005, and in the period from 2007-2009, the number of patients in one region was 3523 persons and for the whole period, more than 4000 patients for Q fever were found.During the epidemic, in 60% of patients the disease was found in asymptomatic form, 20% of patients were hospitalized and 7 people died (13).In the procedure of revelation the epizootiological background, abortions in goats were found in that region.It was proved that the goats were a source of infection.situation in cows and sheep.Vaccination is a measure which will prevent the shedding of Coxiella and reduce the risk from spreading rickettsia, what will significantly reduce the risk of infection for humans.This does not mean that during the next year there will be no infections in humans, the disease can be eliminated but not eradicated (2,13).
The aim of vaccination after the lambing season is to prevent new infections during the next lambing in young animals which are not pregnant.A two time vaccination before pregnancy reduces the risk from C.burnetii infection, majority of abortions are prevented and shedding of C.burnetii after the abortion would be reduced.
According to the literature data, a study on chemiotherapy application was done in infected animals.A possible recommendation for the prevention is usage of tetracycline in sheep, in dose 8mg/1kg, in water, few weeks before lambing (3,5).
General preventive measures that can be applied are the following: prohibition of movement, separation of the animals, prohibition of use of milk and wool, adequate hygiene measures, pest control, wearing of protective clothes, disinfection of equipment and vehicles, removal of placenta parts, adequate removal of manure (storage for 90 days in a roofed storage, afterwards it can be spread on the soil and plow), quarantine when calving and lambing and trade, avoid common grazing.

PREVENTIVE MEASURES
Control of Q fever in domestic animals and prevention of environment contamination with C.burnetii are the most important measures in protection of humans from infection (13).Other measures that can significantly reduce the risk from infection are: education of the residents in rural regions about the ways of infection, possible risks and precautions, education of farmers and other professionally exposed people with the aim of establishing a good agricultural practice.These actions can have influence on the reduction of risk for Q fever.Measures of self protection and zoo hygienic measures: -Personal hygiene during and after working with animals -Work clothing and shoes -Use of protective gloves for the removal of postpartum products -Additional equipment for self protection (mask, goggles) when in contact with materials with high risk (after abortions, during Q fever epidemics, when cleaning the objects for sheep, goat and cattle) -Safe removal of placenta, aborted and stillbirth animals The following procedures were applied (7): -all pregnant animals were removed from all infected farms -all animals were examined with a serological ELISA test three times -bulk samples of milk were examined by PCR method two times -elimination of infected animals -animals were vaccinated according to the age and size of the herd -prohibited visitors -prohibited use of milk -application of biosafety measures During this period 35000 pregnant animals were killed and costs were estimated for 6 million Euros.At the same period of time in neighboring countries Germany and Belgium, Q fever was found in humans and animals (13).
During the year 2010, European agency for food safety (EFSA) had an assignment to come out with measures for the estimation of risk factors and estimation of efficiency of measures for Q fever control in EU (13).
In Barcelona, 2011, a Second European meeting on Q fever was held where new scientific knowledge was discussed about epidemiology, pathogenesis, diagnostic methods and control measures for this important zoonozes.
Data in Europe after the epidemic in Holland show great variability in laboratory diagnostic methods that were used, criteria for explaining the results depending on the aim of diagnosis -herd screening, identification of animal that is shedding rickettsia, epidemiological research, and routine diagnostics.One of the conclusions of the Symposium was that professional knowledge is still insufficient, especially in epidemiology, identification of the ways of infection and of potential reservoirs.Regular veterinary surveillance of animals is necessary in order to track the infection and propose an accurate diagnosis in time.Based on the knowledge we have today, a key measure in Q fever control is vaccination of the animals with C.burnetii vaccine, phase I.

AKNOWLEDGMENTS
The presented work is part of the research done in the project TR31084 and 31071 granted by the Serbian Ministry of Education and Science  , 2001).The name of the virus, arteritis, is based on the characteristic pathogenic activity observed in blood vessels.The virus replicates in muscle cells and endothelial cells of small blood vessels, predominantly arteries, and may lead to the range of inflammatory processes.
According to data from the literature (Holyoak et al, 2008), the disease has been reported in horse populations in all continents: North and South America, Europe, Asia, Australia and Africa.Only Iceland and Japan are considered EVA-free.A research that dates back to the 60ies and 70ies of the last century has established the following seroprevalence rates of EAV: Switzerland 11.3%, England 2.3%, Netherlands 14% and Germany 1.8%.
However, the investigation dated 1998 revealed seroprevalence rates of some 20% in Germany and about 2% in non-vaccinated horses in the U.S.A. (Holyoak et al 2008).In Austria, the EAV seroprevalence ranges between 10 and 12%; however, on some stud farms the rate reaches even 100% (Nowotny 2001).Seroprevalence rate in Anatolia (central Turkey) is 23.4% (Bulut et al.

2012).
So far, the rate of EAV seroprevalence in the Republic of Serbia has not been confirmed, but the results reported by several authors (Petrović et al., 2002; Urošević et al., 2003;Lazić, 2013) indicated the presence of EAV infection in the country.In the Republic of Serbia, the research studies on EVA infection has been conducted only since the beginning of this century, i.e. in 2002, encompassing only imported horses in quarantines.More recently, the disease has been gaining more attention and numerous horse breeders have put efforts to initiate serological testing, foremost of breeding animals.
Infected horses shed the virus in nasal and ocular discharge, urine, aborted foetuses and sperm (stallions).The virus can cause a range of health disorders in horses, including interstitial pneumonia, panvasculitis with consequent oedema predominantly in eye lids, abdomen, distal parts of the limbs, preputium and scrotum, thrombosis associated with bleeding, necrosis in lymph nodes and kidneys, abortions and inflammation of accessory sex glands.Sometimes, the infection may pass without developing any health status disorders (Del Piero, 2000).Infected stallions are potential natural reservoir of the virus.If the virus affects accessory sex glands, stallions remain infected for life and frequently shed the virus in the semen (Del Piero, 2000;Nowotny, 2001;Holyoak et al 2008).Testing the possibility of virus spreading through the semen and its detection in the semen was the topic of interest of numerous researches.Development of modern laboratory diagnostic methods, such as molecular biology

INTRODUCTION
Equine viral arteritis (EVA) is an infectious disease that can affect all ungulate species, as well as all horse categories.The disease is quite common worldwide causing severe health problems and considerable economic losses.The causal agent of the disease is the virus (Equine arteritis virus-EAV) from the family Arteriviridae.EAV is an enveloped, spherical virus of 50-70 nanometres (nm) in diameter possessing single-stranded ribonucleic acid (RNA)

RESULTS AND DISCUSSION
To provide a clear overview of our findings, the obtained results are presented in Tables.The present study encompassed 102 horses (averagely one fourth) out of the total of 355 horses (in 2012) and 386 horses (in 2013) that were raised in 5 examined horse stables (Table 1.).Presence of anti-EAV antibodies was obtained in animals from one horse stable (E), where 62 and 72 horses were raised during 2012 and 2013, respectively.At this farm, 27 horses were examined during the two aforementioned years.Presence of anti-EAV antibodies was confirmed in 16 (59.26%)examined horses in 2012 and in 17 (62.96%)examined horses in 2013.Equine arteritis virus infection has been established only on farm E. Thus, the analysis of the results obtained on this stud farm can illustrate the effects of this infection on the health status of horses, incidence of the infection within different horse categories as well as the potential for virus dissemination on the horse farm.assays, gave rise to more intense research in this field, which have confirmed that seropositive stallions shed the EAV in their semen (Ramina et al., 1999;Balasuriya et al., 2002;Mankoč et al., 2002;Guthrie 2003;Chenchev et al., 2010)

Samples
Blood samples were collected in five horse stables from the territory of Vojvodina Province.The horse stables were designated with letters A -E.The sampling encompassed the same horse population and was performed twice at one-year interval, i.e. in 2012 and 2013.Samples were collected by venipuncture of the Vena jugularis into Vacutainer tubes, and serum separation was performed after spontaneous coagulation.Anamnestic data on health status during the past 3-year period were recorded for each horse subjected to blood sampling.Thus, 102 blood samples of horses were obtained in 2012, and the same number of samples in 2013 so, in total 204 blood serum samples from five horse stables were collected and examined on anti-EAV antibodies presence.
The breed structure of horses at the examined farms was rather diverse, yet several breeds were dominant at particular farms: Lipizzan breed at farms A and E, English pureblood and half-blood breed at farms B and C and Lipizzan and English pureblood and half-blood as well as Nonius breed at farm D. At all investigated stud farms, the breeding systems were in accordance with common practices of horse farming.The stallions are housed in individual boxes, as well as highly pregnant mares or pospartum mares.Other horse categories are raised in groups.Throughout the year, the horses from all farms participate at various competitions, such as galopp races or parade competitions.

Serum samples analysis
Identification of anti-EAV antibodies in blood serum samples was performed by the method of virus neutralization, according to procedure set out by OIE Terrestrial Manual, Chapter 2.5.10,2008 Assessment of seropositive results to EAV on stud farm E according to horse category (Table 3.) reveals an increase in number of seropositive animals in the category of mares, which, according to the obtained anamnesis data, most probably results from mating with seropositive stallions.EAV antibody titre value in the mare marked 31-03459 was 7 log 2 in 2013, whereas the same animal was seronegative in 2012 (Table 2.).This mare was mated by a seropositive shedder stallion during mating season 2012/2013, thus supporting the suspicion of sexual transmission of this viral infection in the stable.Sexual transmission and dissemination of the infection in stud farm E can be better understood by analyzing the distribution of established anti-EAV antibody titre values as well as the mating schedule records.Mating of seropositive stallions during several seasons results in persistence and dissemination According to anamnestic data obtained from staff veterinarians and stud farm manager, during the past 3-and 4-year periods clinical symptoms indicating equine viral arteritis were not observed.Abortions were recorded only sporadically and were mainly associated with twin-embryo pregnancies.Frequently, mares from other stables were introduced to the stud farm for breeding, mostly lacking the documents of serological testing for sexually transmitted infectious agents.
The EVA antibody titre values established during 2012 and 2013 in all investigated horses are presented in Table 2.The analysis of the results indicate virus circulation within the stable, and that the most probable route of virus spread was sexual transmission.Analysis of mating schedule revealed that seropositive stallions were used for mating, therefore an increased number of mares become infected with this virus.The number of seropositive horses increased for one animal in 2013, i.e. from 16 seropositive animals in 2012 to 17 seropositive animals in 2013.The established antibody titre values did not vary significantly; however, the average EAV antibody titre value were somewhat higher in 2013 (3.15 log 2 ) as compared to 2012 (3.11 log 2 ) indicating permanent circulation of the virus on this farm.Still, the seroprevalence to EAV dropped from 25.81% in 2012 to 23.61% in 2013, which is due to the increased number of horses (10 animals) on farm E in 2013.

CONCLUSIONS
Out of the 5 horse stables with in total 355 horses (2012) and 386 horses (2013) encompassed by this research, EAV infection was confirmed at one stable.In that seropositive stable the established antibody titre values did not vary significantly; however, the average EAV antibody titre value were somewhat higher in 2013 (3.15 log 2 ) as compared to 2012 (3.11 log 2 ) indicating permanent circulation of the virus on this farm.According to the obtained results, we may conclude that sexual transmission was the route of virus transmission and dissemination in this stud farm.

ACKNOWLEDGEMENT
The presented work is part of the research done in the framework of the projects TR31084 and TR31071 granted by the Ministry of Education and Science of the Republic of Serbia LITERATURA However, despite positive anti-EAV antibody findings, analysis of general health status of both stallions and mares during 2012 and 2013 revealed neither health status disorders nor failed fertilizations and abortions.The foaling occurred within normal time period, and foals did not manifest any health disorders.These data indicate that EAV infection can persist without health disorders manifestation, which is in accordance with the data reported by other authors (Del Piero, 2000; Urošević et al., 2003;Lazić et al., 2013).
Analysis of the results obtained in this research revealed that they well activity in the presence of organic matters and hard water, not to be toxic or low-toxic for people and animals, not to have an unpleasant smell, not to damage the decontaminated objects, to be proof at storage, accessible for transporting and application, and also economic when exposed in the environment (3,4).
In addition, during the preparation of a disinfectant, some hygienic terms are required.The disinfectant must not show a negative effect on animals and auxiliary personnel in the concentrations recommended for application.The preparation of the disinfectant must be without negative consequences and with no allergic or cumulative properties expressed (5).
For the elimination of causative agent for tuberculosis many facilities are offered in an external environment, but they do not answer to the requirements.Also, not enough of the bactericidal properties of the infectious agent for tuberculosis are studied so far (6).Therefore there was a requirement for the improvement of existing and for the development of more effective devices for disinfection (8).

MATERIAL AND METHODS.
The purpose of our researches was, to study a bactericidal action in new disinfectant, in relation to Mycobacterium.The study was done in SEC "Institute of experimental and clinical veterinary medicine", department of study on brucellosis and tuberculosis.Laboratory research was done according to the methodical recommendations "The order of testing of new disinfectants for veterinary practice" (7).
The first stage of study was on bactericidal properties in potential disinfected with atypical Mycobacteria, type of M. fortuitum № 122.For this purpose the preparations were done by the following "Javelle-Kleyd" and "Geksadekon" instructions.
The exposure of bactericidal properties to atypical Mycobacteria were done in the study with the analysis of bactericidal action of the disinfectant to the causative agent of tuberculosis, bovine kind.The test was carried out with the laboratory culture strain of Vallee.Biological research was performed according to the «Settings on diagnostics of tuberculosis of animals and birds» ratified on 26.05.1997 (9).
The estimation of the results gained during the study on bactericidal properties of disinfectant was done based on the results of crop and biological research.

INTRODUCTION
One of the main directions in the system of prophylaxis and fight against tuberculosis of animals belongs to the disinfection.
Disinfection is a complex of measures, aiming on elimination of causative agents of infectious diseases of man and animals in an environment, namely: disinfection of the second link of epizootic chain -factors of transmission.These measures enable the destruction of pathogenic microorganisms in the environment, in order to break the epizootic chain and stop development of epizootic process (1).Disinfection should be planned as a part of the prophylactic and epizootic measures (2).
For each disinfectant, it is important to have the following: good solubility in water, wide spectrum of antimicrobial action, not to reduce antimicrobial Based on the information given in Table 2, it can be seen that the preparation "Javelle-Kleyd" in concentration 0,1 % and exposure of 30 minutes and 1 hour and also "Geksadekon" in concentration 3 % and exposure of 5 and 24 hours inactivate Mycobacterium tuberculosis of bovine kind, within the laboratory conditions.The growth of test culture M.bovis was noticed in control test tubes with nourishing environment provided at that time.For confirmation of positive results for crop method, the experiment on laboratory animals was performed.Research results on guinea-pigs are given in Table 3.

RESULTS
Results which are presented in Table 1 verify that analysis with different modes of disinfectant "Javelle-Kleyd" are the following: -For the concentration of 0,05 % and exposure time 1 hour and -For the concentration of 0,1 % and exposure time 30 minutes, 1 hour, the preparation disinfects atypical Mycobacteria.-With the disinfectant "Geksadekon", the results were the following: -For the concentration of 3 % and exposure time 3 hours and 24 hours, the preparation disinfects atypical Mycobacteria.
Therefore, as next step, we conducted research with the test culture of bovine kind.The results of this exposure are given in Table 2. Based on the information given in Table 3, it can be seen that in the guineapigs from experimental groups, in the internal organs there were no characteristic changes related to tuberculosis changes that could be found.On the other hand, in the control group, in lost (?) laboratory animals, pathological changes were discovered, that can be characterized as typical for tuberculosis.It should also be marked, that when allergic test for tuberculosis was performed in guinea-pigs, the intradermal reactions to tuberculin were discovered in the animals from the control group.

CONCLUSION
1. Crop and biological methods have bactericidal activity in relation to Mycobacteria tuberculosis, bovine kind with disinfectants "Javelle-Kleyd" in concentration 0,1 % and exposure of 30 minutes and 1 hour and "Geksadekon" in concentration 3 % and exposure time of 5 hours.2. "Javelle-kleyd" and "Geksadekon" disinfectants in certain modes, are possible to apply for prophylactic and forced disinfection in happy and unhappy economies in relation to tuberculosis of animals.

Sample collection
Location: Uvac River canyon valley with its three lakes (Uvačko, Zlatarsko and Radoinjsko Lake) as a Special Nature Reserve is protected natural asset of great importance according to the Decree of the Government of the Republic of Serbia ("Official Gazette of the RS" No. 25/06 and 110/06).It occupies some 7 500 ha between mountain Zlatar massif in the Southwest and mountain Javor in the Northeast.The estimated population of griffon vultures in this reservation is currently around 100 nesting couples, and total population (including young birds) is some 300 birds.The adult nesting birds mostly do not leave their natural habitat, whereas young birds migrate during winter months to the north (via Slovenia and Italy to the Alps), to the south (Bulgaria, Greece, Israel and Africa) and to the west (France and Spain).

Results AND Discussion
In the '90s of the last century, the griffon vulture population in the Republic of Serbia was threatened by extinction.In the territory of Uvac-lakes, there were only seven individuals of this rare species from the Accipitridae family.The survival of this endangered species was enabled through continual delivery of slaughterhouse waste (some 50 tons per year).Nowadays, Uvac is the largest colony of this unique bird species in the Balkans, and among the large ones in Europe.Diet based on animal carcasses, i.e. sheep, bovine and horses from intensive farming systems, undoubtedly contributed to the survival of griffon vultures; however, feeding slaughterhouse waste could negatively affect the health of avian scavengers and promote transmission and spread of bacteria from these sources (Blanco et al., 2006).
In this research, bacteria of the genus Salmonella were isolated from five (33.3%) fecal samples originating from Eurasian griffon vultures.All isolates fermented glucose, produced H2S and L-lysine decarboxylase.Negative reactions were obtained for Voges Proskauer and indole tests, lactose fermentation, production of β-galactosidase (ONPG) and urease.In the national Reference Laboratory for Salmonella, Shigella, Vibrio cholerae, Yersinia enterocolitica, all isolates were serotyped as Salmonella enterica subsp.enterica ser.Veneziana "The magnificent Uvac valley", natural habitat of Eurasian griffon vulture (http:// www.politika.rs/rubrike/putovanja/U-carstvu-beloglavog-supa.lt.html) Sampling was carried out at Uvac natural reservation in June 2012, during early morning hours.At the moment of sampling, around 50 Eurasian griffon vultures were present at the selected location.According to observation results, the birds were in good health condition.Fifteen fresh fecal samples were collected using sterile swabs.The samples were cooled and transported to the laboratory of clinical microbiology of the Scientific Veterinary Institute "Novi Sad".

Microbiology examination
Isolation of E. coli and Salmonella spp.: Collected fecal swabs were inoculated into 2mL peptone water (Buffered peptone water, CM1049, Oxoid, Basingstoke, UK) and vortexed for 30 sec at maximum rpm.For the isolation of E.coli, 30 μL of suspension was inoculated onto the Columbia blood agar base (CM0331, Oxoid) with 5% sterile defibrinated sheep blood and MacConkey agar (CM0007, Oxoid).Inoculated plates were incubated at 37 o C during 24-48h.Colonies of the characteristic appearance for the Enterobacteriaceae, were subcultivated on nutrient agar (CM0003, Oxoid).The colonies were examined using catalase and oxidase test and biochemical series test: "IMViC" (indole -methyl red-Voges Proskauer-citrate).
ampicillin.The reports on bacteriological examination of samples originating from Eurasian griffon vultures are extremely limited (Milan et al., 2004;Sabočanec et al., 2005;Kocijan et al., 2009), whilst data on antibiotic susceptibility of bacterial isolates are still lacking.
Undoubtedly, humans play an important role in spreading pathogens among the wild bird population through contamination of waters by sewage waste, inadequate waste management and by artificial feeding of birds.Predominance of enteric bacteria and antibiotic-resistant strains within the population of griffon vultures, as well as their role in survival and spread of such bacteria in the natural environment remains unclear.Transmission of bacteria from birds to mammals requires repeated exposure and large numbers of microorganisms (Gerlah, 1994), thus the importance of Eurasian griffon vultures as a source of human and animal infection is not significant, which is due to the "solitary lifestyle" of these birds in inaccessible terrain.However, as feeding ecology is the principal factor that influences exposure of birds to intestinal bacteria (Benskin et al., 2009), the diet of griffon vultures based on slaughterhouse waste might have some negative impact upon health status of these birds and spreading of resistant strains of enteric bacteria over large distances by young migrating birds., 1997;Milan et al., 2004), since their isolation is usually not associated with symptoms of the disease (Palmgren et al., 2006) nor macroscopic or microscopic lesions characteristic for salmonellosis (Milan et al., 2004).Epizootic outbreaks of salmonellosis in raptors are extremely rare as compared to other bird species (Milan et al., 2004).The incidence of one particular serotype indicates the existence of one same infection source or possibility of specific adaptation of Salmonella to the host, when it establishes itself as a part of the intestinal flora (Tizard, 2004).In free living raptors, examination of fecal samples revealed mainly low incidence of salmonellas, being 1.5% (Skov et al., 2008) , 1.9% (Mikaelian et al., 1997) and 4.19% (Reche et al. 2003).Within the framework of large-scale investigation of salmonellosis in wild animals, Milan et al. ( 2004) established a prevalence of 8.5% (7/82) in wild birds, while S. Typhimurium was isolated in one, out of three examined fecal sample originating from Eurasian griffon vultures.

LITERATURA
Bacteriological examination of fecal samples in this research revealed presence of Escherichia coli in four (26.6%) samples.Kocijan et al. (2009) performed bacteriological, mycological and parasitological analysis of fecal samples (n=10) and vomit samples (n=5) of griffon vultures from the island Cres, Croatia.Escherichia coli was the most frequent bacterial isolate in both sample categories (feces 9/10, vomit samples 2/5).In raptors, Escherichia coli can be associated with diverse pathologic lesions (bumble foot, pericarditis, hepatic abscesses, salpingitis, and egg peritonitis) (Morishita et al., 1997).However, the virulence factors of particular E.coli strains, which are of importance in humans and mammals, do not accurately predict the relative importance of the strains for birds (Gerlach, 1994).In this research, one E. coli isolate manifested multiple-resistance to streptomycin, tetracycline, amoxicillin+clavulanic acid, ampicillin and trimethoprim/sulfamethoxazole, whereas two isolates were resistant to been widely studied and is regarded as the most important bacterium causing "aeromonosis or haemorrhagic septicaemia or motile aeromonas septicaemia" in fish (Rhaman et al. 2001) and other aquatic animals (Hill et al. 2010;Pearson et al.2000).There have been a number of epidemiological studies indicating Aeromonas species as a cause of diarrheal disease in children, elderly people and immunocompromised patients (Figueras, 2005;von Gravaenitz, 2007).
The widespread of the bacteria in the aquatic environment and the stress caused by intensive breeding are predisposing factors for the disease.Stressful environmental factors, especially high water temperature, high levels of ammonia and nitrite, sudden changes in pH, and low concentrations of oxygen increases the possibility of disease occurence (Jeremic et al. 2005).
Several extracellular products (ECP) of A. hydrophila are considered important virulence factors, primarily aerolysin, extracellular lipase, cytolytic enterotoxin, hemolytic toxin and extracellular protease (John et al., 1997, Shome et al., 2005).Detection of virulence genes by PCR is very useful for the identification of pathogenic isolates of aeromonads (Uzbas et al., 2000).
Detection of aerolysin (aerA) using PCR and RFLP is considered a reliable for identification of virulent strains of A. hydrophila (Kingombe et al., 1999).The aim of this study was to determine the presence of aerA gene in A. hydrophila strains isolated from carp with motile aeromonad septicaemia.

MATERIAL AND METHODS
Five samples of the one-year-old carp with clinical symptoms of bacterial infection were used for isolation.From each fish, samples were collected from the kidney, liver, spleen and gills.The samples were streaked on tryptic soy agar (TSA, HiMedia), Mueller-Hinton agar (HiMedia) containing 5% defibrinated sheep blood erythrocytes (BA), selective Rimler-Shotts (RS) media (HiMedia), and incubated at 30 o C for 24 to 48 h .Following incubation, one typical colony (entire circular, convex, white to greyish, semitranslucent, size 2 to 3 mm, haemolytic) was selected from each plate with a pure culture and subcultivated in order to test the purity of isolates.The isolates were preliminary grouped according to colony morphology, haemolysis, and pigmentation before they were stored at -80 o C in 15% glycerol until further characterization.The type strain of Aeromonas hydrophila ATCC 7966 (American Type Culture Collection) was included in the phenotypic characterization.
Isolates were classified as Aeromonas hydrophila according to their reactions in the API 20E (Biomerieux) and following conventional tests, based on standard bacterial taxonomic procedures (Holt et

INTRODUCTION
Aeromonas hydrophila, a Gram-negative, motile rod that is a member of the family Aeromonadaceae (Joseph & Carnahan 2000;Abbott et al. 2003), has Pure cultures of Aeromonas hydrophila were obtained from all samples (Figure 2). Figure 2. Growth of Aeromonas hydrophila on Rimler-Shotts medium.
For identification of virulence genes, five colonies of A. hydrophila were selected and 24 hours old cultures were used for extraction of genomic DNA.DNA extraction was performed using commercial kit (QIAamp DNA Mini Kit, Qiagen) according to manufacturer's protocol.Detection of the aerolysin gene was performed using polymerase chain reaction (PCR) (Chu et al., 2005).Primers were used to detect 462bp aerolysin gene fragment (Aero1: 5'-CTCAGTCCGTGCGACCGACT-3' and Aero2: 5'-GATCTCCAGCCTCAGGCCTT-3').Amplification was performed by 35 cycles of denaturation at 95 o C for 1 min, annealing at 56 o C for 2 min and extension at 72 o C for 2 min.After amplification, PCR products were characterized by 1.5% agarose gel electrophoresis in Tris-borate-EDTA buffer.

RESULTS
On a carp pond, localized in the northeastern part of the Republic of Serbia, in the spring, during a sudden increase in water temperature, increased mortality of young carp occured.The main signs of the diseased fish were anorexia, exophthalmus, redding due to haemorrhage of the skin and swimming at the surface of the pond, near fresh water supply.In scaled fish, scale pockets become edematous, causing lepidorthosis (Figure 1).Internal organs were edematous with hemorrhage and erythema on liver and kidney.most important symptoms are exophthalmos, skin redness and fluid collection in the scale pockets (Faktorovich, 1969).
There is evidence that the motile Aeromonas complex involves secondary and opportunistic pathogens, but ability of A. hydrophila to cause disease and death of fish should not be overlooked because occasionally highly virulent strains emerge.Regardless of whether or not the organism serves as a primary or secondary invader of stressed fish, it is often the final insult that leads to death (Plumb and Hanson 2011).
In attempt to explain the pathogenesis of infection caused by A. hydrophila several virulence factors were investigated.Toxins with haemolytic, cytotoxic and enterotoxic activities have been described in many Aeromonas spp.(Chopra et al., 1990).
Although rare, A. hydrophila may cause high mortality among cultured fish without presence of severe external (stressful) influences.This inconsistency may result from the presence of A. hydrophila strains that possess specific virulent or pathogenic characteristics (Plumb and Hanson 2011).
In our study, PCR was performed to detect aerolysin (aerA) gene as a genetic marker for the determination of virulence.Role of aerolysin (aerA) gene in the pathogenicity of Aeromonas genus was previously demonstrated (Kozaki et al., 1989, Shaw, 2003).In present study, PCR amplification of A.hydrophila isolates for the 426-bp aerA gene, in samples from diseased fish, showed the presence aerolysin gene, which is an essential A. hydrophila virulence gene.Similar results were obtained in previous studies with A. jandaei (Chacón, 2003).It is well known that the screening of specific cytotoxin and hemolysin genes is the most effective way of detecting and characterizing Aeromonas virulence factors (Yousr et al., 2007).

CONCLUSIONS
Despite disagreement among scientists regarding significance of A. hydrophila infection, the frequency of its appearance in aquaculture environment, together with a high potential for stress, shows that this problem sholud not be ignored, because it is often what kills the fish.PCR test for the detection of aerolysin gene proved to be a useful tool for the detection of virulent strains of Aeromonas hydrophila.

ACKNOWLEDGEMENT
This study was financially supported by a grant from the Ministry of Education and Science of the Republic of Serbia, under the project TR31075, TR-31011.
A PCR amplification revealed that all A. hydrophila isolates were PCR positive for the aerA gene.

RESULTS
Table 1.shows the prevalence estimates provided by blood serum sample collected from boars imported from Denmark.The sera from 120 boars, originating from 3 different farrow-to-finish herds, were examined by ELISA test for the presence of Salmonella antibodies.After the import, boars were placed in quarantine.After a period of resting from transport, within 5 days from the due date, blood samples were taken from boars for serological testing.All of the tested blood serum samples taken from boars during the quarantine in 2011 and 2012 were negative for the presence of antibodies specific for Salmonella (Table 1).
A year after the first blood sampling for serological testing, blood serum samples were taken again from the same boars.

INTRODUCTION
Salmonella spp.does not normally cause clinical disease, but subclinical Salmonella spp.infections constitute an important food safety problem throughout the world (Kranker et. al., 2003).Salmonella spp.transmission in swine herds is a significant health and hygiene issue concerning public health and food safety.The major source of Salmonella contamination in pork is fattening the pigs from latently infected herds (Beloeil et al., 2004).In spite of the widely acknowledged value of controlling Salmonella in the live animal reservoir, and copious research endeavors, there is still much to learn about the control of Salmonella pre-harvest, as well as discerning the most cost-effective approaches to approaching control in the pork chain (Gorton et al., 2000).Salmonella IgG antibodies have been demonstrated in blood serum from boars using commercial kits, because these antibodies persist long after the infection (Šišak et al., 2011).The presence of antibodies indicates that the pigs were exposed to the enteric pathogen in a period of development, but on the other hand, the time needed for seroconversion suggests that pigs are carriers of Salmonella while still seronegative, and also different immune responses can affect the serological tests (Boyen et al., 2008).
Import of boars to Serbia occurs regularly in recent decades.When importing boars from breeding stock and/or countries free of certain diseases, there is a chance to confirm the presence of latent infections by certain diseases.The objectives of this study were to: examine Salmonella seroprevalence in boars from 3 farrow-to-finish herds, imported from Denmark.Boars were imported twice, in 2011 and 2012.

MATERIAL AND METHODS
Selection of herds.Three Serbian farrow-to-finish swine herds with a capacity of 2000 sows, with an intensive way of keeping the pigs were selected for the study.Each farm imported 20 boars from Denmark in 2011 and 2012.Boars spent 30 days in quarantine, and after that were forwarded for exploitation.All the boars had tags on their ears and a tattooed number.Artificial insemination has been performed on examined farms.
Sampling and laboratory analysis.Before leaving the quarantine (within 5 days from the due date), blood samples were taken from all boars for testing.After a year, blood samples were again taken from the same boars.Blood was taken by the puncture of the brachiocephalic plexus of the boars.A blood serum sample from each boar was frozen, and blood serum (harvested after

DISCUSSION
This study provided a unique opportunity to compare serological status of boars prior to arrival and after spending a year in Serbia.All boars were seronegative upon arrival to Serbia.Boars were located on three different farms, where after a year their serological status should represent an indicator of Salmonella spp.presence on monitored farms.After one year from import of 60 boars in 2011, 11 boars were found seropositive, whereas on one farm they were all seronegative.The farm where all boars were seronegative after one year, conducted high-level biosecurity measures during that year, boars were kept in a separate building, had no contact with other animals, as well as people who worked with them.After one year from import of boars in 2012, on the same farm there was 6/20 seropositive boars.On the farm during this period isolated Salmonella from feed and as a result of the findings of six seropositive boars.Feed as a source of infection for pigs and the prevalence of Salmonella in feed have been investigated by Molla et al., 2010.While the other two investigated farms had not high levels of biosecurity measures.According to the literature data, herd seroprevalence rates for Salmonella are 93% in Germany, 59% in Denmark, 79% in Greece and 72% in Sweden, evaluated at test cut-off of OD%>10, and herd cut-off of 1 or more seropositive animals.The average rate within herd seroprevalence was 24% for Germany, 9% for Denmark, 14% for Greece and 10% for Sweden (Lo Fo Wong, 2001).Our study found higher seroprevalence in 5/6 herds; interestingly, the same herd that was seronegative became positive in the next year.The average seroprevalence within herd was 24.17, which is also a higher level compared to research results of other authors (Nowak et. al., 2007;Bonde and Sørensen 2012).

CONCLUSION
Based on the results the survey to be concluded that in Serbia there is a high seroprevalence of Salmonella.This suggests the necessity of more intensive implementation of biosecurity measures on farms, as well as control of feed for pigs, which can be a potential source of various infections.The high seroprevalence of Salmonella in pigs Serbia is a potential risk of contamination of the meat, and therefore infection in human.
Ključne reči: Leptospiroza, metodi dijagnostike, L. interrogans serovar hardjo were confirmed in 67 and 18 samples, respectively.Initial ELISA-positive finding was observed on day 15, showing increasing tendency throughout the monitoring period and reaching its maximum value on day 42.Method of blood sample cultivation resulted in isolation of L. interrogans serovar hardjo in 33 (18.03%) on day 3 at the earliest, whilst highest isolation rate was observed on day 17 post challenge.Applying polymerase chain reaction (PCR) method, genome or genome sequences of L. interrogans serovar hardjo were detected in 67 (56.30%) out of 119 blood serum samples.PCR method revealed positive finding as early as on day 1 post challenge, whereas the highest rate of positive findings was recorded on day 19.Comparison of the results obtained by methods of cultivation and PCR during the period from experimental day 1 to 21, i.e. period prior to administration of chemotherapeutic agents, demonstrated high level of linear correlation of r = 0.8105 at the 0.01 significance level.After dihydrostreptomycin therapy administered from day 21 post infection, L. interrogans serovar hardjo could not be isolated using the method of blood sample cultivation.Contrary to that, PCR method revealed the presence of L. interrogans serovar hardjo genome in 23

INTRODUCTION
Most cases of L. interrogans serotype hardjo infection in animals are asymptomatic, yet associated with shedding of leptospires in the environment via the urine.Identification of infected animals, particularly carriers, is a multifaceted problem in the view of validity of applied serological tests as well as highly complex and time-consuming isolation procedure.Keeping up-to-date with modern diagnostic techniques for leptospirosis as well as continuous improvement of laboratory diagnostic methods resulted in abundant knowledge on the nature and consequences of this infection and its importance in both human and veterinary medicine.In that respect, development and introduction of novel diagnostic tests and procedures have become the paramount issue in the diagnostics of leptospirosis and related infectious diseases.Thus, the goal of this research was to investigate the application of diverse laboratory methods and to evaluate their validity in the diagnostics of leptospirosis.Immunological assays are highly applicable in the diagnostics of leptospirosis and thus increasingly used in laboratory practice.Reaction of microscopic agglutination (MA), being an eligible diagnostic method, has been the most widely used serological test so far.In the recent years, ELISA has become widely used.However, all the aforementioned tests demonstrated some limitations and drawbacks regarding sensitivity and specificity in the detection of subclinical infections in animals.While the method of isolation is the most accurate and reliable diagnostic tool yet highly demanding, time-consuming and expensive, the polymerase chain reaction (PCR) is sufficiently sensitive, specific and rapid method enabling detection of even small number of leptospires.Capacity of polymerase chain reaction (PCR) to detect presence of 5-10 bacteria per ml of the sample indicates its high sensitivity, which makes it more appropriate and applicable as compared to the method of cultivation Smith et al. (1994).

MATERIAL AND METHODS
Population of 11 rabbits were challenged with live culture of L. interrogans serovar hardjo by skin scarification procedure.Blood and blood serum samples of challenged animals were collected every other day throughout the 3-week period (i.e. until day 21), and then once weekly during following five weeks.Blood sera were tested for the presence of L. interrogans serovar hardjo specific antibodies applying the method of microscopic agglutination (MA) test with live seven-day-old cultures of reference leptospiral serotypes (Royal Tropical Institute Amsterdam, The Nederland) as well as modified ELISA for detection of L. hardjo-specific antibodies in blood serum samples of infected rabbits.In this assay, basic components of the commercial bovine kit were used (IgG and IgM, producer: Central Diagnostic, Lelystad, the Netherlands A).The modification has implicated replacement of the conjugate with anti-rabbit immunoglobulins (IgG, IgA, IgM) produced in pig and conjugated with peroxidase (MP Biomedicals, Inc; Aurora, Ohio SAD, cat.No 1135).The titre of used immunoglobulins was determined by chessboard titration.The working titre of conjugated immunoglobulins was 1:200.Detection of L. hardjo in blood serum was performed by the method of cultivation in liquid medium (by Johnson) supplemented with bovine albumin and 200 µg/ml 5 fluorouracil (5FU) and by polymerase chain reaction (PCR).The reaction was done according to adapted protocol for isolation and diagnostics of leptospiral DNA described by Merien et al. (1992).Isolation of DNA from urine samples was performed according to QIamp ® DNA Mini and Blood Mini Kit (Quiagen ® , Germany) protocol for DNA purification from blood and body fluids (spin-protocol).PCR reaction was accomplished using two oligonucleotide primers described by Mrerien et al. (1992), which correspond to nucleotides 38-57 A Lepto (5'-GGC GGC GCG TCT TAA ACA TG-3') and B Lepto (5'-TTC CCC CCA TTG AGC AAG ATT-3') from the primary structure of L. interrogans serotype canicola strain Moulton 16S rRNA gene.The resulting specific product was 331 bp in size.DNA amplification was performed in a 25 μl reaction, using a thermal cycler "MultiGene Thermal Cycler, TC9600-G" manufactured by LabnetInternational, Inc (NY., USA).Temperature regimen encompassed 15-minute Taq polymerase activation period at 95 0 C with simultaneous phase of initial denaturation of DNA template from the sample, followed by 35 cycles encompassing 1.5-minute denaturation at 94 o C, 1-minute hybridization (annealing) at 63 o C, 2-minute elongation at 72 o C and final elongation (extension) at 72 o C during 10 minutes.The resulting amplification product was applied onto the 1.5% gel supplemented with ethidiumbromid and subjected to electrophoresis.The results were captured and interpreted using the illuminator.

RESULTS AND DISCUSSION
The results of the detection of specific antibodies against L. hardjo in rabbits using dark field microscopic agglutination (MA) are displayed in Table 1 and Chart 1.The results pertain to the period from the start of the experiment until day 56, i.e. the experimental period is divided into two phases.The first phase encompassed sampling from day 1 to day 21, whereas second phase encompasses weakly sampling throughout 5-week period after administration of chemotherapeutic agents.78 Comparison of GMT values obtained by MT and ELISA tests revealed the following: samples examined by MA test manifested increased levels of specific antibodies, which exceeded the boundary level of 1:100 (GMT titre 4 log2/ 25 ) as early as on day 7.The level of L. hardjo specific antibodies diagnosed by MA method increases until reaching the maximum level on days 13 and 15, and then shows decreasing tendency and drops below the boundary positivity level on day 42 (Cole J.R. Jr,et al.1973).Contrary to MA method, ELI-SA demonstrated considerably longer period (15 days) between the infection and exceeding the boundary positivity levels being 45% (PP) or (GMT titre 4 log2/ 25 ).During the further course of the experiment, GMT values manifested permanent increasing tendency until day 42, when they reached the maximum levels, and then started to drop down gradually.It is to be emphasized that MA method enables seropositivity diagnosis from day 7 to day 42 postinfection.In ELISA, the time span of diagnosis extends from day 15 to day 56, i.e. to the end of the investigation period (Chart 3).Legend: (-) negative (+) positive Ø -died rabbits Chart 2. Distribution of GMT antibodies against L. hardjo in blood sera of rabbits challenged by skin scarification -diagnosed by ELISA period underwent certain modifications aimed at improving the reliability of ELISA method (Cole et al., 197;, Márcia Costa Ooteman et al.,2006).The articles of Bercovich Z. et al. (1990) and Levett P.N. (2002) confirmed the ability of this method to detect specific antibodies in the serum of infected animals at the earliest between the day 5 and day 10 post-challenge, which corresponds with our results.The method of cultivation of leptospires is highly reliable; however, it manifests some considerable limitations such as poor stability of leptospires and their rapid decay in the environment, time-consuming isolation procedure as well as high level of biohazard for the personnel.The method of polymerase chain reaction (PCR) enables detection of leptospiral infection at a very early stage thus increasing the chance for prompt and successful therapy.This is of particular importance in latent human and animal infections and their suppression and prevention.Data from the available literature pertaining to comparison of the results obtained by PCR and other methods strongly indicated its high sensitivity as compared to other methods used so far in the diagnostics of leptospirosis (Gravekamp et al., 1993;Grgić Ž., 2011, Doctoral Dissertation).

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Graph 1 -
Incidence of Q fever in Vojvodina during the period from 1966-2012* * Institute for public health of Vojvodina: Infectiousdiseases in AP Vojvodina, 2012.

Figure 1 .
Figure 1.The pathological symptoms of the common carp suffering from motile aeromonad septicemia.

Chart 3 .
Distribution of GMT values for antibodies against L. hardjo in blood sera of rabbits challenged by skin scarification -diagnosed by MA test and ELISA Testing of correlation levels of GMT values for the applied methods ( i.e.MA and ELISA) in the population of rabbits challenged by skin scarification revealed weak inverse linear correlation of r = -0.3277with 0.05 level of Headings in the paper are: Introduction, Material and Methods, Results, Discussion (or Results and Discussion), Conclusion and Literature.Introduction points on the most important, i.e. most recent data regarding the topic with a short presentation of the aims of this research.

Table 1 .
Overview of examined horses according to the farm and EVA antibody finding

.
Virus neutralization was performed by using rabbit-kidney cell line RK-13 and Bucyrus strain of EAV.According to the OIE Terrestrial Manual, antibody titre values ≥2 log 2 are considered as seropositive result.Animals that do not have a certified history of vaccination against EVA are considered infected, i.e. such stallions are considered potential source of infection.

Table 3 .
Number of horses with positive anti-EAV antibody finding on farm E in 2012 and 2013, according to horse categories

Table 2 .
Overview of anti-EAV antibody finding in horse blood sera on farm E in 2012 and 2013

Table 5 .
Distribution of the established anti-EAV antibody titre values according to horse categories in stud farm E in 2012 * -negative antibody finding

Table 2 .
Crop method of determination of bactericidal properties in "Javelle-Kleyd" and "Geksadekon" disinfectants, in relation to the causative agent of tuberculosis, bovine type /strain of Vallee/ Legend: "+" -growth of Mycobacteria is present; "-" growth of Mycobacteria is not present.

Table 1 .
Results of research of bactericidal action of antimicrobials in crops, related to Legend: "+" -growth of Mycobacteria is present; "-" growth of Mycobacteria is not present.
1* e-mail: dubravka@niv.ns.ac.rsArchives of Veterinary Medicine, Vol. 6, No. 2, 45 -53, 2013.Milanov D. i dr.: Enteric bacteria … aimed at prevention of Bovine Spongiform Encephalopathy.Limitation of natural habitat and reduced availability of their natural prey in our country resulted in changes of feeding habits of Eurasian griffon vultures.The diet of these wild birds is based mainly on slaughterhouse waste from intensive farming systems.Such diet could contribute to alterations of common microflora of the digestive tract.The available literature data on bacteriological examination Eurasian griffon vulture samples are still sparse both in the territory of Balkans and at a global level (Milan et al., 2004; Sabočanec et al., 2005; Kocijan et al., 2009).The aim of our study was to investigate presence of enteric bacteria in fecal samples (Escherichia coli and Salmonella spp.), which are not considered as a part of common intestinal microflora of Eurasian griffon vultures, and to examine the antibiotic susceptibility of these bacterial isolates.
Kruse et al., 2004) in the epidemiology of salmonellosis is still debated, several studies have confirmed the possibility that wild birds could serve as the source of human infection (Millan et al., 2004,Kruse et al., 2004).Eurasian griffon vultures (Gyps fulvus) are at the top of the food chain and playIsolates that resulted +/+/-/on the "IMViC" test were further examined using BBL Crystal Enteric/Nonfermenter test (Becton Dickinson, Detroit, MI, USA).
(Pavoković, 2005), 2005)ffon vulture population is closely associated with abandoning of extensive livestock breeding in the area and thus food scarcity, poisoning(Sabočanec et al., 2005)and laying poisoned baits for large predators(Pavoković, 2005)as well as the EU Regulations on mandatory carcass disposal (Bruner et al., 1945)ulder R., Scott G.W.: Wild birds and human pathogens in the context of ringing and migration.Ringing & Migration,23, 193-200, 2007.2.Benskin C.M., Wilson K., Jones K., Hartley I.R.: Bacterial pathogens in wild birds: a review of the frequency and effects of infection.11:i:e,n,x).This Salmonella serotype was first isolated from an apparently normal Italian civilian food handler in Venice, Italy, and described as a new serotype in 1945(Bruner et al., 1945).S. Venezianae is not listed among the 15 most frequently isolated serotypes in Serbia (www.who.int/gfn).According to the available literature, data on isolation of this serotype are scanty.In the period 1998-2008 in the U.S., only one case of laboratory-confirmed isolation of this serotype in humans was reported (CDC, 2008).S. Venezianae was isolated from mesenteric lymph nodes of one horse out of 500 examined samples taken from 100 slaughtered healthy horses(Bonardi et al., 1997).Disc diffusion test with S. Veneziana isolates did not reveal resistance against antibiotics used in the experiment.The birds are generally considered as healthy carriers of Salmonella (Mikaelian et al.
1* e-mail: stojanac.n@gmail.com66thawing) was examined for specific anti-bodies against Salmonella spp.using an indirect ELISA.Samples with an OD% > 10 were considered seropositive.Data analysis.Data were entered into an Excel spreadsheet (Microsoft Excel 2010) and imported into Stata (Stata 8 Intercooled for Windows 9x) in which data were analyzed.Descriptive analysis was done in MiniTab version 14 (MiniTabR14b) and Excel (Microsoft Excel 2010).

Table 1 .
Salmonella spp.prevalence estimates provided by blood serum sample collected from boars imported from Denmark

Table 2 .
Finding of antibodies against L. hardjo in blood serum of rabbits challenged by skin scarification -ELISA (expressed in %)