SEROEPIDEMIOLOGICAL INVESTIGATION OF MYCOPLASMA BOVIS IN CALVES

Within the multifactorial etiology of respiratory infections in cattle, Mycoplasmae play an important role. Most of the Mycoplasma microorganisms play a minor role in the development of the infections in cattle, contrary to Mycoplasma bovis (M. bovis) that is commonly the primary agent of the disease. Respiratory tract mucosa is the primary colonization site of M. bovis in cattle. Colonization of the upper respiratory tract of calves with M.bovis oft en occurs within the fi rst few weeks of life. Animals with chronic infection and without clinical symptoms occasionally shed M. bovis and they are highly important for the epidemiology of the infection. Stress factors such as transportation, entrance into the feeding object, coldness, etc. are associated with the secretion of M. bovis from a nose. Diagnostic procedure relies on clinical symptoms and detection of causative agent, regardless of whether the infection is found in individual animals or in the entire herd. Serological detection of M.bovis antibodies is oft en a reliable diagnostic method. Th e most used indirect method is ELISA test. During a two-year period, blood serum samples from calves (beef cattle) were examined and analyzed. Calves originated from cattle farms (big and small) of HolsteinFriesian and Simmental breed. Th e total number of 3777 samples was examined applying ELISA (Biovet Inc. Mycoplasma bovis Antibody Test Kit Bovichek® M.bovis). Positive fi ndings were obtained in 182 1* PhD Branka Vidić, principal research fellow; PhD Sara Savić, research fellow, PhD Živoslav Grgic, research fellow, Nadežda Prica, spec.chem., Scientifi c Veterinary Institute, Novi Sad, Rumenacki put 20, 21000 Novi Sad, Serbia, branka@niv.ns.ac.rs PhD Ljiljana Suvajdžić, professor, Faculty of Medicine, Department of Pharmacy, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbia Archives of Veterinary Medicine, Vol. 7, No. 1, 3-9, 2014. Vidić B. et al.: Seroepidemiological investigation ...


INTRODUCTION
Mycoplasmas is recognized as the cause of some of the most severe and economically most costly diseases in cattle ( Gonzalez et al, 1993;Gonzalez and Wilson 2003). More than 20 different Mycoplasma species have been isolated from cattle with different clinical symptoms of a disease (Henderson and Ball 1999). Most of the Mycoplasma microorganisms have a secondary role in cattle infection, contrary to Mycoplasma bovis (M. bovis), which has a primary role in the occurrence of the disease. M.bovis was fi rst isolated back in 1961 in the US, as a disease with clinical symptoms of severe mastitis in cattle (Hale et al, 1962), and then during the next 40 years, it has spread to Europe, Asia and the rest of the world (Filioussis et al, 2007). So far, the presence of mycoplasmosis in cattle has been reported inmost European countries. The prevalence of M.bovis is underestimated and other bacteria are often isolated in calves with pneumonia or cattle with mastitis, where actually M.bovis is the primary cause. Only a few laboratories in the world routinely perform the monitoring of mycoplasmas. The occurrence of M.bovis in the herd is always associated with the cases of pneumonia, mastitis and arthritis (Pfutzner and Sachse, 1996). As compared to economic losses from respiratory diseases, the losses associated with mycoplasmosis in the cattle industry of US or UK are very high, tending to increase due to mycoplasmatic mastitis cases (Rosengarten and Citti, 1999).
M.bovis is widely spread among bovine population in enzootically infected areas. Th e infection is usually introduced into the new herds by clinically healthy calves or young cattle shedding the causative agents. Infected cattle shed mycoplasmas via the respiratory tract for many months or years representing the permanent reservoair of the infection. Respiratory tract mucosa is the primary site for the colonisation of M. bovis in cattle. Respiratory tract mucosa and mammary gland are the most important locations for the maintenance and secretion of M. bovis, which can persist even several months. Stress factors such as transportation, entrance into the feeding object, coldness, etc. are asso- Diagnostic procedure relies on clinical symptoms and detection of causative agent, regardless of whether the infection is found in individual animals or in the entire herd. ELISA is the mostly used indirect method. Serological detection of M.bovis antibodies is oft en a highly reliable diagnostic method. Th e level of antibodies detected by ELISA method persists for many months, especially in case of preceding month-long antibiotic therapy at herd level. In such cases, the isolation of the agent is very diffi cult. All other serological tests, such as possible indirect haemagglutination or fi lm inhibition, are not as successful as indirect ELISA and thus not widely used. Commercial diagnostic tests are available in the market used worldwide.

MATERIALS AND METHOD
During a two-year period, blood serum samples from calves (beef cattle) were collected, examined and analyzed. Th e samples were taken from animals originating and living in diff erent regions in the territory of Serbia. Th e calves of Holstein-Friesian and Simmental breed originated from several cattle farms (big and small. Th e total number of 3777 samples was collected and examined. Th e diagnostic was performed using ELISA method. Th e diagnostic kit used in this research was a commercial Biovet Inc. Mycoplasma bovis Antibody Test Kit Bovichek ® M.bovis, which is used in a routine laboratory work. Th e blood sera were analyzed using indirect ELISA test according to manufacturer's instructions. ELISA (Enzyme -linked immunosorbent assay) is a sensitive and specifi c method for detection of specifi c antibodies against certain infectious agent from blood sera. Antibodies from the serum bind with the antigen contained in a layer coating the wells of the test and an antigenantibody complex is formed. Subsequently, the complex is stained to enable better visualisation.

RESULTS AND DISCUSSION
In total, 3777 blood serum samples of calves from diff erent farms were analyzed for the presence of specifi c antibodies against Mycoplasma bovis. Th e analysis was performed using indirect ELISA method, a commercial kit. Positive fi ndings were detected in 182 animals, i.e. 4.81% of the total population examined.
Th e fi nding of specifi c antibodies against Mycoplasma bovis is presented in Table 1. Similar results were obtained in another study performed in Serbia that included diff erent regions and diff erent diagnostic laboratory. In this research, 2.74% of calves proved positive to specifi c antibodies against Mycoplasma bovis in an indirect ELISA method (Vojinovic et al, 2012).
Mycoplasmae play an important role in the multifactorial etiology of respiratory infections in cattle. Several Mycoplasma spp. can cause severe mastitis in cattle, but M.bovis is the the predominant one. Th e disease spreads rapidly, that is, many cows manifest clinical signs of mastitis in one or more udder quarters in a very short period. In lactating cows, the infection mostly aff ects the entire udder. On farms with history of cattle mycoplasmosis, problems with joints, reproductive failures, pneumonia in calves and respiratory problems in adult cattle were recorded (Stokka et al, 2001). Dairy cows with mycoplasmatic mastitis show a drastic drop of milk production. Considering the infectious nature of the disease, clinical symptoms spread within the herd very fast, thus appropriate control measures have to be implemented (Vidic et al, 2012).
Unlike the majority of bacterial infections, the therapy of Mycoplasma infections is highly demanding, which is due to organism's resistance to mostly used antibiotics. Vaccination is a potential strategy to control M.bovis infection; however, the eff orts to develop eff ective vaccine for use in young calves have been problematic so far. An eff ective program for the control of M. bovis infections includes a number of factors such as early detection of carriers and their removal from the heard, appropriate vaccination schedule, breeding conditions providing minimum environmental stress, housing with good air circulation, "all in all out" management practice to prevent infection transmission from older animals to younger ones or at least separating the calves from

AKNOWLEGMENTS
Th e presented work is a part of the research done in the project TR31071, granted by the Serbian Ministry of Education and Science.