LAST SURVEY OF MYCOPLASMA BOVIS PREVALENCE IN POLISH CATTLE AFFECTED WITH RESPIRATORY SYNDROME

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1 Bull Vet Inst Pulawy 56, , 2012 DOI: /v LAST SURVEY OF MYCOPLASMA BOVIS PREVALENCE IN POLISH CATTLE AFFECTED WITH RESPIRATORY SYNDROME KATARZYNA DUDEK AND DARIUSZ BEDNAREK Department of Cattle and Sheep Diseases, National Veterinary Research Institute, Pulawy, Poland Received: August 17, 2012 Accepted: December 6, 2012 Abstract The aim of the study was to determine the prevalence of Mycoplasma bovis infection in clinical cases of bovine respiratory disease (BRD) in There were examined 841 serum samples and 41 nasal swabs and lungs of cattle from different farms of six regions of Poland (13 provinces). The obtained results indicated that the mean prevalence of M. bovis infection in Polish cattle population suffering from BRD was 64.3% and among them 8.4% of the animals were highly positive. On the other hand, in particular regions, it altered % in east region, 52.7% in central region, 66.4% in south region, 64.1% in north-west region, 56.5% in north region, and 58.8% in south-west region. It should be added that within the presented regions, the highest values (82.4%) were reported in south region (Malopolskie province), whereas the lowest ones (42.9%) were in east region (Lubelskie province). However, field strains of M. bovis were isolated from the lungs of affected cattle only in the north-west region of Poland (Zachodniopomorskie province). Key words: cattle, Mycoplasma bovis, bovine respiratory disease. Mycoplasma bovis is a major bacterial infectious agent causing mycoplasmal infection and chronic diseases such as pneumonia, arthritis, mastitis, keratoconjunctivitis, and otitis, in cattle (14). Fourmonth-old calves and feedlot animals are particularly predisposed to pneumonia caused by M. bovis infection (1, 14, 19). The bacterium colonisation of the upper respiratory tract of calves was also observed earlier in few-week-old animals (5). The colonisation is facilitated through the presence of different virulence factors of the bacteria such as variable surface membrane proteins determining strong structural and antigenic variation among different strains of M. bovis and facilitating to avoid the host defence (4). Affected animals are able to shed the bacteria from the respiratory tract for many months or years, which enhances the possibility of cross-infection (14, 16). There are well known nonspecific clinical signs of M. bovis infection and its chronic nature as a result of the long-term presence of the pathogen inside lesions to probable later releasing and reinfection (18). This specific nature of M. bovis infection may be also the result of the isolate-dependent ability of the pathogen to impair a production of oxygen radicals by bovine leukocytes and inhibit their phagocytic activity (8, 25). M. bovis is able to survive in a host organism or environment mainly by the ability to the formation of a biofilm (14), which may enhance the pathogen resistance to antibiotics, phagocytes, or antibodies (13). M. bovis is able to modulate the immune system of host. Its suppressive effect on blood cells results from the induction of lymphocyte apoptosis and inhibiting different function of some leukocytes, i.e. lymphocyte proliferation, neutrophil degranulation, and others (10, 21, 22, 24). The destructive effect on immune system may initiate lung lesions as it was reported by Vanden Bush et al. (23). Under conditions of natural infection, M. bovis can also stimulate acute phase response (APR) in cattle. In this case, a significant increase in characteristic acute phase protein concentrations such as SAA and Hp was observed (6). M. bovis is also considered to be an effective proinflammatory agent in infected cattle, what is manifested by an increase in eicosanoid production (PGE 2, PGF 2α, TXB 2 ) as a result of arachidonic acid cascade stimulation (7). Currently, M. bovis is also perceived as a primary infectious agent in a multifactorial aetiology of bovine respiratory disease (BRD) in both health and economic aspects (15). In the pathogenesis of BRD, M. bovis plays the same role like Pasteurella multocida, Mannheimia haemolytica, and some bovine respiratory viruses (BVDV, PI3) (9, 11, 15). The difficulties connected with the isolation and identification of M. bovis infection under the field

2 448 conditions and its antibiotic resistance reduces significantly the control of the infection (15). Moreover, a lack of available commercial vaccines in Europe against M. bovis infection additionally accounts for the regular monitoring of the infection in cattle herds. High seroprevalence of Mycoplasma bovis infection in Polish cattle population, reported during the last four years, motivates a further epidemiological study on this subject, especially on cattle affected with BRD. Material and Methods Animals, sample collection, and assays. Totally, 841 bovine sera and 41 tissue samples (nasal swabs, lungs) were collected from cattle with clinical sings of BRD (fever, nasal discharge, cough, dyspnoea, apathy, lack of appetite) during The cattle were kept in individual pens (up to 20 animals) of different farms from six regions of Poland (13 provinces). The blood samples were collected from the vena jugularis externa and then centrifuged to obtain the sera to further analysis. The nasal swabs were sampled from the nasalpharyngeal cavity using sterile tubes with transport medium, whereas tissue samples were collected from bovine carcasses with detectable lung lesions characteristic for mucosal haemorrhagic lung inflammation. Before the analysis, the serum samples, swabs, and tissue material were stored under freeze conditions (-20 ºC). The following numbers of the samples were examined: east region 239 serum samples (four provinces) and 11 nasal swabs (one province), central region 146 serum samples (two provinces), two lung samples (one province), and 22 nasal swabs (one province), south region 140 serum samples (two provinces), north-west region 259 serum samples (three provinces), four lung samples (one province), and two nasal swabs (one province), north region 23 serum samples (one province), and southwest region 34 serum samples (one province). The presence of M. bovis antibodies in sera with an evaluation of positiveness degree (from 0 to 4+) was carried out with the use of an indirect ELISA (BioXDiagnostics), whereas the isolation and identification of M. bovis antigen from nasal swabs and lung samples were performed with the use of an antigenic direct ELISA (Pulmotest, BioXDiagnostics). Positive/negative results were calculated according to the manufacturer s instructions. The evaluation of positiveness degree was conducted by Quality control sheet attached to the method instruction of indirect ELISA. Results The administrative division of Poland into regions and provinces is presented in Fig. 1. The analysis of M. bovis antibodies in sera of cattle from east region of Poland indicated 172 positive samples of which 27 were determined as high positive ( 2+ of positiveness degree; Table 1). From total number of 239 serum samples examined in this region, the percentage of seropositive animals was 72.0 of which high positive samples came to On the other hand, no M. bovis was isolated from nasal swabs (Table 2). In central region of Poland, the percentage of positive sera was 52.7 among 146 examined samples of which seven (4.8%) samples were highly positive (Table 1). The analysis of nasal swabs and lungs did not reveal the presence of M. bovis antigen (Table 2). From total of 140 sera examined in south region, 66.4% (93 samples) of positive results were obtained, of which high positive sera constituted 3.6% (Table 1). The serological analysis of affected cattle deriving from north-west region of Poland indicated 64.1% of seropositive animals and among them 11.6% (30 sera) were highly positive (Table 1). It should be emphasised that in highly positive animals, the positive isolation of M. bovis was obtained also from the lungs (Table 2). The serological survey of cattle in north region of Poland, based on the evaluation only of sera from Kujawsko-Pomorskie province, gave 56.5% of positive results. Similarly, in south-west region with Opolskie province, the same analysis revealed 58.8% of positive samples of which 5.8% was highly positive (Table 1). Discussion The study, carried out in 2011, on the presence of specific antibodies against M. bovis in the population of Polish cattle suffering from BRD deriving from five regions of Poland indicated that 64.3% of all affected animals was positive and among them almost 8.5% was highly positive ( 2+ of positiveness degree). On the other hand, in individual regions it altered: east region 72.0%, central region 52.7%, south region 66.4%, north-west region 64.1%, north region 56.5%, and south-west region 58.8%. The highest percentage of positive sera among all examined regions of Poland was observed in east region (71.97) of which maximum value was obtained for Podlaskie province (78.0). However, the highest percentage of positive sera among all provinces was in Malopolskie province (south region) and it constituted The results are in the agreement with the previous study on the evaluation of M. bovis antibody in cattle serum performed in Poland between 2007 and 2010 (3). The study showed that the mean percentage of seropositive animals was on the average level of 76.65, regardless the clinical status of the animals. The analysis was performed on total of 3,670 cattle sera collected from 16 provinces of Poland. Seropositive cattle came from 292 herds within a total number of 361 ones. Interestingly enough, 2,529 animals originated from respiratory disease cases of which 864 cattle were affected with BRD. The analysis of M. bovis seroprevalence in the latter case indicated that 73.15% of positive animals came from 76 herds representing 89.41% of the examined ones. The greatest

3 449 number of serum samples in BRD cases was tested in Podlaskie province of which 68.4% was seropositive. However, the highest percentage of positive samples was observed in Malopolskie province (88.9), whereas the lowest percentage of the affected animals characterised Pomorskie province (60.0) (3). The minimum percentage of seropositive animals among provinces was observed in Lubelskie province and it amounted to 42.9, whereas the same analysis performed among the regions of Poland indicated that the lowest percentage of seropositive animals was observed in central region and it constituted On the other hand, the analysis only with regard to highly positive sera in different regions of Poland indicated that its maximum value was observed in northwest region of Poland (11.6%). The same analysis concerning provinces of the region showed the highest positive results in Zachodniopomorskie province (17.9%). Table 1 Seroprevalence of M. bovis infection in Polish cattle affected with BRD in different regions/provinces of Poland Region Province Total number of samples Number of seronegative samples (%) Number of seropositive samples with degree of positiveness from 1+ to 4+ (%) Total number of seropositive samples (%) East Lubelskie 7 4 (57.1) 3 (42.9) (42.9) Podkarpackie (32) 61 (59.2) 9 (8.7) (68) Podlaskie (22) 76 (64.4) 13 (11) 2 (1.7) 1 (0.8) 92 (78) Swietokrzyskie 11 4 (36.4) 5 (45.5) 2 (18.2) (63.6) Total (28) 145 (60.7) 24 (10) 2 (0.8) 1 (0.4) 172 (72) Central Lodzkie (50) 12 (37.5) 3 (9.4) 1 (3.1) - 16 (50) Mazowieckie (46.5) 58 (50.9) 2 (1.8) 1 (0.9) - 61 (53.5) Total (47.3) 70 (47.9) 5 (3.4) 2 (1.4) - 77 (52.7) South Malopolskie Slaskie (17.6) 54 (79.4) 2 (2.9) (82.4) (48.6) 34 (47.2) 3 (4.2) (51.4) Total (33.6) 88 (62.9) 5 (3.6) (66.4) Lubuskie Wielkopolskie (34.8) 67 (59.8) 6 (5.4) (65.2) (35.2) 53 (49.1) 15 (13.9) 2 (1.9) - 70 (64.8) North Total (35.9) 136 (52.5) 28 (10.8) 2 (0.8) (64.1) Kujawskopomorskie (43.5) 13 (56.5) (56.5) Total (43.5) 13 (56.5) (56.5) Southwest Zachodniopomorskie (41.0) 16 (41.0) 7 (17.9) (59.0) Northwest Opolskie (41.2) 18 (52.9) 1 (2.9) - 1 (2.9) 20 (58.8) Total (41.2) 18 (52.9) 1 (2.9) - 1 (2.9) 20 (58.8) Total values for regions/provinces (35.7) 470 (55.9) 63 (7.5) 6 (0.7) 2 (0.2) 541 (64.3)

4 450 Table 2 Isolation of M. bovis from nasal swabs and lungs of cattle affected with BRD in different regions/provinces of Poland Region Province Sample type Number of examined samples Number of samples with the presence/lack of M. bovis antigen (+/-) + - nasal swab 2-2 East Lubelskie nasal swab 2-2 nasal swab 7-7 Total lungs 1-1 Central Mazowieckie lungs 1-1 nasal swab Total lungs 2-2 North-west Zachodniopomorskie nasal swab 2-2 lungs Total All regions/provinces North-west region East region Central region South region North region South-west region Fig. 1. Administrative division of Poland into regions and provinces. The results of the study differ considerably from the same data published in the literature. For instance, during the five-year similar study carried out in Great Britain, the seroprevalence of M. bovis infection in different age groups of cattle averaged 22% per year of total of 8,959 examined serum samples (2). The last serological studies performed in Switzerland indicated the percentage of M. bovis infection was 50.03, whereas in other European countries some seropositiveness in cattle averaged between 10% and 20% in France (12) and 11% in Hungary (20). On the other hand, in Northern Italy, the percentage of seropositive cattle came to even 88 of which 100% was observed in calves and 76% in adult cattle from a total number of 140 examined animals (17). The examination of nasal swabs and lung samples collected from affected animals allowed to isolate one field strain of M. bovis from cattle lungs in Zachodniopomorskie province. It is worth to remind that, there was also observed the highest number of positive sera. M. bovis is a pathogen, whose an effective isolation is very difficult due to its high susceptibility to the environmental factors such as transport and specific requirements during the cultivation Due to the occurring

5 451 problems; it is possible to make certain diagnostic errors by the veterinarians. Despite the lower seroprevalence of M. bovis in Great Britain, there were observed more frequent its isolations from respiratory diseases in cattle (2). From total of 1,413 samples examined during eleven-year study in Great Britain, from 735 M. bovis isolates, 622 came from cattle the respiratory tract. The main mycoplasmal species supporting contamination were Mycoplasma bovirhinis, Mycoplasma canis, and Acholeplasma laidlawii (2). For comparison in France, the M. bovis was detected in 106 bronchoalveolar lavage samples from a total number of 135 (78.5%) examined. In 81.1% of positive samples there was observed the coinfection with other respiratory pathogens (1). The present results are in agreement with the previous study concerning the seroprevalence of M bovis infection in cattle affected with BRD and indicated that the percentage of positive animals is similar during the last years (3). Previously and currently it has been reported that the level of the seroprevalence in affected cattle oscillated about 69%. On the other hand, the analysis of province level indicated that the high M. bovis seroprevalence (about 80%) was noted in Malopolskie province through the same time. Considering the lack of commercial vaccines against M. bovis infections in Europe and an increasing resistance of isolated strains to most antibiotics used in veterinary medicine in cattle, it seems to be relevant a regular monitoring of mycoplasmal infections in Poland, especially in cattle affected with BRD. References 1. Arcangioli M.-A., Duet A., Meyer G., Dernburg A., Bézille P., Poumarat F., Le Grand D.: The role of Mycoplasma bovis in bovine respiratory disease outbreaks in veal calf feedlots. 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