COMPLEX OF RESPIRATORY DISEASES IN CATTLE FROM THE ASPECT OF PARAINFLUENCA 3 VIRUS

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1 Biotechnology in Animal Husbandry 25 (5-6), p , 2009 ISSN Publisher: Institute for Animal Husbandry, Belgrade-Zemun UDC COMPLEX OF RESPIRATORY DISEASES IN CATTLE FROM THE ASPECT OF PARAINFLUENCA 3 VIRUS S. Lazić 1, T. Petrović 1, D. Bugarski 1, N. Kendrišić 2 1 Scientific Veterinary Institute «Novi Sad», Novi Sad, Republic of Serbia 2 Veterinary Clinic «Kendrišić», Šid, Republic of Serbia Corresponding author: lazić@niv.ns.ac.rs Original scientific paper Abstract: Respiratory diseases of cattle are considered to be the most frequent causes of health problems in now days cattle industry. Among all respiratory diseases the Bovine Respiratory Disease Complex BRDC is the most significant. BRDC is manifested with severe inflammation of respiratory tract and lungs. The disease is caused by viruses, bacteria and stress factors. The major etiological agents of this disease are, however, the viruses especially accompanied with stress. Among many viruses the most significant role in BRDC is attributed to herpes virus type-1 (BHV-1), parainfluneca-3 virus (PI-3), the bovine respiratory syncytial virus (BRSV), bovine viral diarrhoea virus (BVDV) while less important are bovine adenoviruses and corona viruses. BRDC is common in our cattle industry mostly in intensive rearing of beef cattle. Ethiopathogenesis, clinical and pathological characteristics of BRDC are presented in our literature usually from the aspect of BHV-1 and BVDV infections. A small number of papers deal with the disease from the aspect of PI-3 and BRSV. Therefore the goal of this paper is to present BRDC outbreak, accompanied with PI-3 infection, in one bull calves fattening unit of industrial type (number of bull calves in one production cycle is 800 to 1200). The paper presents clinical signs, procedure for PI-3 virus isolation and bacteria detection in diseased beef cattle. In the paper is also presented the immune response in 30 beef cattle (from the stable where the infection occurred) on PI-3 virus. The immune response was determined through detection of specific antibodies to PI-3 at the time of occurrence of the disease and also at 3 and 6 weeks after diseases outbreak as well as at the end of fattening, i.e. 4 months after PI-3 infection. The geometric mean value of antibody titre against this virus was at the moment of disease occurrence 14.58, while in 3 and 6 weeks after outbreak it was and 54.44, and at the end of fattening it was cattle Key words: BRDC, PI-3 virus, specific antibodies against PI-3 virus, beef

2 704 S. Lazić et al. Introduction Pathogenesis of bovine respiratory syndrome presents the combination of stress factors that disturb the defence mechanisms of host especially respiratory organs accompanied with the influence of infectious agents (usually one or more viruses or bacteria). The viral infection reduces defence mechanism of respiratory organs and enables colonization of lower parts of respiratory system i.e. diaphragmatic lobes of lung with bacteria that are usually present in nasopharyngeal region. Therefore, respiratory syndrome is multi-causal and because of that, term "bovine respiratory disease complex BRCD" was accepted (Dyer, 1982). Diagnosis can be established based on clinical symptoms such as depression, fever (body temperature of 40 o C and higher), loss of appetite, cough, dyspnoea with or without nasal secret, even if etiological agent is not recognized. However, it is better to perform laboratory test and determine etiological agent. This will provide better success in therapy and easier establishing of general prophylaxis and vaccination. The therapy is based on antibiotic treatment and helps in recovery, but since BRDC is accompanied with loss of weight and increased feed conversion, this disease results in poor income. For preventive reason vaccination against various bacteria and viruses that contribute to BRDC development is usually conducted. However, data show that only 75% of vaccinated animals are actually protected even when vaccinated with best vaccines. For this reason much effort is directed to examination of the causal agents, its genotype and phenotype modifications, pathogenesis and immunological process during BRDC (Hodgins et al., 2002). The dominant factors that predispose BRDC are breeding conditions and stress. Therefore, control and preventive measures include taking care of housing and feeding, from the beginning at neonatal period until the end of fattening. Additionally, overcrowding must be avoided, good ventilation provided without drift, stress must be reduced as much as possible, manipulation with animals should be reduced etc. (Bryson, 1996). The viruses that most frequently play the role in BRDC are: bovine respiratory syncytial virus (BRSV), bovine infectious rhinotracheitis (IBR - BHV- 1), parainfluenza-3 virus (PI-3) and bovine viral diarrhoea virus (BVDV) (Bryson, 1996). Other viruses, such as bovine adenovirus (BAdV), bovine coronavirus (BCV) or bovine calicivirus, bovine parvovirus, bovine herpesvirus type 4 (BHV- 4), bovine reovirus and rhinovirus can be isolated in certain cases of BRDC although their role has not been established yet and to this end these agents are mostly causing episode infections during BRDC. Bacteria usually present in mucosa of respiratory tract, especially in nasopharyngeal region, also play a significant role in ethiopathogenesis of BRDC. The most common are bacteria from the family Pastaurellaceae, especially Mannchemia haemolytica biotype A

3 Complex of respiratory diseases 705 and Pasteurella multocida (Frank and Briggs, 1992), so the diseases is frequently diagnosed as lung pasteurelosis. Additionally, Histophilus somni, Arcanobacterium pyogenes and Mycoplasma species such as M. mycoides, M. dispar, M. bovis etc. can be involved in BRDC (Petrović et al., 2008). The virus PI-3 was found in acute and chronicle cases of pneumonia in cattle. The disease is most frequently occurred in young animals. Clinically, fever, caught, mucous yellow nasal and eye discharge can be seen in mild course of the disease (Gale, 1970; Laegreid et al., 1989; Richey, 2002). The infection occurs most frequently during fall and winter. It is worthy to mention that PI-3 virus is often present in mix infections with BHV1 and BVDV and in that case the clinical outcome is severe. Infection with PI-3 virus presents the frequent predisposing factor for development of bacterial pneumonia as a consequence of virus influence on phagocyte cells and its function (Gale, 1970; Laegreid et al., 1989; Richey 2002; Hadgins et al., 2002). The infection of alveolar macrophages with PI-3 virus in vivo and in vitro causes lower ability to neutralize and ingest bacteria including inhibition of phagosome-lizocine function and enhanced production of metabolites of arachidonic acid that inhibits other phagocyte functions (Laegreid et al., 1989). Experimentally it was proven that 6 days post infection with PI-3 virus alveolar macrophages from bronchial discharge showed lower expression of Fc and C3b receptors. This induced reduced phagocytes of opsonised bacteria (Brown and Ananaba, 1988). Besides that, macrophages and monocyte, infected with PI-3, depress lymphocyte proliferation introduced by mitogens in vitro as well as response of lymphocytes to Concavalin A and interleukin 2 (IL-2) (Basaraba et al., 1994). Lymphocytes from peripheral blood show enhanced cytotoxic activities against cells infected with PI-3 virus between 5 and 9 days post infection. Alveolar macrophages and lymphocytes are often infected with the virus, so defence against other pathological agents directed to respiratory tract is lower. The infection with PI-3 virus has influence on activity of the smooth muscle in lungs. Folkerts et al. (1990) determined that contraction of the smooth muscle was enhanced 45% after histamine induction, while the number of inflammatory cells was 1.5 time higher than in healthy animals. It was noted also that after experimental infection with PI- 3 virus distributed by aerosol, there was enhanced release of histamine from mastocite. The most effective measure in prevention of PI-3 infection is vaccination. It could be firmly concluded that in human, as well in veterinary medicine, no other procedure is so effective in reducing the disease and lethal effect and with high success in the improvement of animal and human health like various vaccination strategies are (Rogan and Babiuk, 2005). As refers to PI-3, there are many vaccines that can be divided in two basic groups: inactivated vaccines and attenuated vaccines. However, not many vaccines have only PI-3 virus or its antigens, usually vaccines are made from the combinations of various agents that cause respiratory syndrome.

4 706 S. Lazić et al. It should be emphasized that in our country the occurrence of BRDC from the aspect of virus etiology was mostly narrowed to bovine herpes virus type 1 and bovine viral diarrhoea virus. Much has been highlighted related to various aspects of infection, control, diagnostic, eradication as well as prevalence of BHV-1 and BVDV (Lazić et al., 2008; Petrović et al., 2008). In our literature there are only few data on PI-3 virus as the causative agent of BRDC, especially in beef cattle. The goal of this paper is to present the most important facts related to respiratory symptoms in breeding cattle caused by PI-3. The procedure of virus isolation, as well as immunological reactivity of cattle to PI-3 from the onset of the disease to the end of fattening is shown and also the bacteria found during the outbreak are presented. Materials and Methods Animals. Respiratory syndrome, caused by PI-3, was investigated on one beef cattle farm of industrial type of rearing. The capacity of this farm was about 1200 animals. The disease was noted in one stable where 120 animals were held in 4 boxes. Thirty animals from one box were examined where severe symptoms were recorded and the largest number of animals was sick. From 5 most affected animals nasal swabs were taken to establish etiological diagnosis and from all animals blood samples were collected on few occasions. The examined animals were approximately the same age (6 months) and weight (300 kg). All the animals were female and of the same domestic breed (type domesticated Simmental). Nasal swabs examination. Nasal swabs were collected from 5 animals to determine bacteria in aerobic and microaerofil conditions. On Petri dishes with blood agar (5% of sheep red blood cells) each swab was streaked next to Staphylococcus aureus. The plates were incubated three days on 37 o C and each day examined for the bacteria growth. From the same animals the swabs was taken for virology examination. For virus isolation, the swabs were transported in a media (medium for cell culture growth) to the Laboratory of Virology and subsequently prepared for inoculation on cell culture. Blood samples. Blood sampling was done in 30 heifers by puncture of vena jugulars at multiple time points, i.e. at the time when the disease occurred at 3 and 6 weeks after the first symptoms and at the end of fattening, i.e. almost 4 months after the disease stopped. The sera were stored at -20 o C until examination for specific antibodies against PI-3 virus. Determining specific antibodies against PI-3. Determination of the specific antibodies titre against PI-3 was done by haemagglutination inhibition test (HI test). To determine antibody titre in HI test the reference PI-3 virus, the virus strain SF-4 (National Veterinary Services Laboratories, Ames, Iowa, USA) with 4 haemagglutination units were used. The test was run in microtiter plates with U

5 Complex of respiratory diseases 707 bottom. In these plates first double dilutions of sera was done and the same amount of the working solution of the virus was added to each well. After 30 minutes of incubation to the mixture serum-virus, the suspension of 1% of erythrocyte of guinea pig was added. The test was read after incubation of 45 minutes, i.e. after haemagglutination control was clearly visible. The highest dilution of sera that completely inhibited the haemagglutination activity of the virus was the end point in titration. Each plate included control of working solution of the reference virus and the control of monospecific reference antiserum (Ašanin et al., 2006). Results and Discussion Clinical signs in diseased heifers. The disease started 3 to 4 days after different actions (the animals were transferred from one to another box, individually marked, prepared for immunoprophylaxis against anaerobic infections). Clinical symptoms of respiratory syndrome were the most prominent in one box where 50% of animals were sick (this was the highest number of diseased animals comparing to other boxes). In sick animals the temperature was from 40 to 41 o C, with dyspnoea, while in most heifers cough was severe. From nostrils serious discharge was visible, and after 2 to 3 days became slimy, but not purulent. The mucus of nasal cavity was mildly hyperemic and all animals experienced loss of appetite while at the same time apathy was absent. This clinical report and anamnesis data point on PI-3 viral infection, so the etiological diagnostic was focused on this virus. Results of bacteriological examination. From the nasal swabs in all diseased animals, among clinically important bacteria, Pseudomonas aeruginosa and Nocardia species were found, while in one swab Pasteurella multocida was isolated. Isolation and identification of PI-3 virus. The content of each swabs from nasal mucosa, taken from 5 diseased heifers, after the samples had been prepared for virology examination, were separately inoculated to Madin Darby Bovine Kidney (MDBK) cells. In the first blind passage, that lasted 7 days, no visible lesions due to virus could be detected. At the third day of second blind passage from the content of the swab from animal marked 4061 typical PI-3 virus lesions in cell culture were found. Additional examination with the monospecific sera against PI-3 virus confirmed the PI-3 virus isolation. In this way the virus was diagnosed in sick animals after respiratory syndrome had been noticed. The disease was further confirmed by detection of specific antibodies to PI-3 virus. According to serology data presented in Table 1, it can be seen that the highest antibody titre to this virus was found 6 weeks after infection. The geometric mean titre at this time point was and it was higher than geometric mean titre (45,21) obtained 3 weeks from the onset of the disease. Thus "the golden rule was confirmed: parallel (acute and reconvalescent) blood samples collected in

6 708 S. Lazić et al. intervals from 3 to 6 weeks have to be examined. The geometric mean titre of specific antibodies 6 weeks from the onset of the disease was 3 times higher comparing to the time point when the disease appeared. When analyzing specific antibody titre at the time when the disease occurred it can be noticed that most heifers (18) had already been infected with the PI-3 virus. Antibody titre of 1:16, found in some cases, clearly showed that some animals had already been in contact with the virus. Most likely that these animals were subclinically infected, but further manipulation that causes stress, reactivated the virus thus resulting in clinical disease. Table 1. Specific antibody titre value against PI-3 virus in heifers diseased from respiratory syndrome caused by PI-3 virus No. Marks Period when the diseases occurred 3 weeks after the disease occurred Antibody titre 6 weeks after the disease occurred End of fattening :4 1:128 1:64 1: :16 1:4 1:32 1: :8 1:8 1:8 1: :32 1:128 1:128 1: :16 1:64 1:64 1: :16 1:64 1:32 1: :64 1:32 1:32 1: :16 1:32 1:128 1: :32 1:256 1:128 1: :8 1:64 1:16 1: :64 1:32 1:64 1: :4 1:32 1:128 1: :8 1:32 1:64 1: :2 1:8 1:16 1: :4 1:32 1:64 1: :8 1:128 1:64 1: :8 1:64 1:64 1: :16 1:16 1:16 1: :16 1:256 1:256 1: :8 1:16 1:64 1: :256 1:128 1:32 1: :32 1:64 1:32 1: :4 1:16 1:64 1: :16 1:128 1:128 1: :16 1:64 1:64 1: :16 1:64 1:64 1: :32 1:64 1:64 1: :8 1:64 1:64 1: :16 1:64 1:64 1: :64 1:64 1:64 1:32 MGA* titre 14,58 45,25 54,44 22,11 * Mean geometric antibody titre

7 Complex of respiratory diseases 709 Conclusion The results presented here show that PI-3 can be the causative agent of respiratory syndrome in beef cattle and that this virus deserves more attention. The obtained specific antibody titres provide evidence that heifers were immunology reactive, while specific antibodies against PI-3 can persist until the end of fattening. Acknowledgment The research was financed by the Provincial Secretary for Science and Technology Development, project number Kompleks respiratornog oboljenja goveda sa aspekta infekcije parainfluence 3 virusom S. Lazić, T. Petrović, D. Bugarski, N. Kendrišić Rezime Oboljenja organa respiratornog sistema goveda se smatraju najvećim zdravstvenim problemom današnjeg govedarstva. Među mnogobrojnim oboljenjima organa respiratornog sistema "kompleks respiratornog oboljenja goveda" (Bovine Respiratory Disease Complex BRDC) sigurno predstavlja najznačajnije oboljenje. BRDC se manifestuje teškim oblicima zapaljenja disajnih puteva i pluća, izazvanih virusima i bakterijama uz uticaj stresogenih faktora. Ipak se smatra da su glavni etiološki faktori ovog oboljenja virusi uz delovanje faktora stresa. Od mnogobrojnih virusnih agenasa najznačajniju ulogu u nastanku BRDC imaju: goveđi herpesvirus tip-1 (BHV-1), parainfluenca-3 virus (PI-3), goveđi respiratorni sincicijalni virus (BRSV), virus goveđe virusne dijareje (BVDV), dok su drugi virusi, npr. goveđi adeno i korona virus, ipak manjeg značaja. BRDC je česta pojava u govedarstvu u Srbiji, a posebno u sistemima intenzivnog odgoja tovne junadi. Etiopatogeneza, kliničke i patoanatomske karakteristike BRDC su u našoj stručnoj i naučnoj literaturi najčešće predstavljane sa aspekta infekcije BHV-1 i BVDV. Malo je literaturnih podataka koji ovo oboljenje predstavljaju sa aspekta infekcije virusom PI-3 i BRSV. Prema tome, cilj ovoga rada je da se prikaže pojava BRDC u jednom tovilištu junadi, industrijskog tipa, (broj junadi u turnusu iznosi od ) sa aspekta infekcije virusom PI-3. U radu je prikazana klinička slika, postupak izolacije virusa PI-3 i nalaz bakterijske mikroflore kod obolele junadi. Takođe, u radu je prikazan i imuni status

8 710 S. Lazić et al. kod 30 junadi (boks u kojem se pojavila infekcija) na virus PI-3. Imuni status je određivan utvrđivanjem nivoa specifičnih antitela protiv virusa PI-3 u momentu pojave bolesti, 3 i 6 nedelja posle pojave oboljenja i na kraju tova, odnosno 4 meseca posle pojave infekcije PI-3 virusom. Utvrđena geometrijska srednja vrednost titra antitela protiv ovog virusa je iznosila u momentu pojave oboljenja 14,58, tri i šest nedelja posle pojave oboljenja 45,25 i 54,44, a na kraju tova 22,11. References AŠANIN R., KRNJAJIĆ D., MILIĆ N. (2006): Priručnik sa praktičnim vežbama iz mikrobiologije sa imunologijom. Fakultet veterinarske medicine, Beograd BASARABA R.J., LAEGREID W.W., BROWN P.R., SILFLOW R.M., BROWN R.A., LEID R.W. (1994): Cell-to-cell contact not soluble factors mediate suppression of lymphocyte proliferation by bovine parainfluenza virus type 3. Viral Immunology, 7, BRYSON D. (1996): Infectious bovine respiratory disease emerging issues and progress towards control. World Buiatrics Congress - BCVA Edinburg, UK. BROWN T.T., ANANABA G. (1988): Effect on respiratory infections caused by bovine herpes virus-1 and parainfluenza-3 virus on bovine alveolar macrophage function. Am.J.Vet.Res., 49, DYER R.M. (1982): The bovine respiratory disease complex: a complex interaction of host, environment and infectious factors. Compend. Eontin. Educ., 4, FOLKERTS G., JANSSEN M., NIJKAMP F.P. (1990): Parainfluenza-3 induced hyperreactivity of the guinea pig trachea concides with an increased number of bronchoalveolar cells. Br. J. Clin. Pharmacol., 30, FRANK G.H., BRIGGS R.E. (1992): Colonization of the tonsils of calves with Pasteurella haemolytica. Am. J. Vet. Res., 53, GALE C. (1970): Role of parainfluenza-3 in cattle. Journal of Dairy Science, 53, 5, HODGINS D.C., CONLON J.A., SHEWEN E. (2002): Respiratory Viruses and Bacteria an Cattle, In K.A. Brogden and J.M. Guthmiller (ed.), Polymicrobial Diseases, Chapter 12, ASM Press. LAEGREID W.W., LIGGITT H.D., SILFLOW R.M., EVERMANN J.R., TAYLOE S.M., LEID R.W. (1989): Reversal of virus-induced alveolar macrophage bactericidal dysfunction by cyclooxigenase inhibition in vitro. J. Leukoc. Biol., 45, LAZIĆ S., PETROVIĆ T., LUPULOVIĆ D., BUGARSKI D., PUŠIĆ I., POLAČEK V., MALJKOVIĆ M. (2008): Raširenost infekcije herpesvirusom 1 u malim zapatima goveda na području Južnobačkog i Sremskog okruga. Arhiv veterinarske medicine, 1, 1,

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