**2. Diagnosis**

366 A Bird's-Eye View of Veterinary Medicine

infections in field outbreaks of BRD (Kita et al., 1994). The highest specific antibody titres

During the necropsy the anatomicopathologic changes are observed in the ventral part of the lung lobes and involve, in the decreasing the apical, cardiac and cranial part of the caudal lobes. The area can involved 5 - 40% of the lung tissue. Histologically, the changes concerning intensive accumulations of lymphocytes in the peribronchiolar tissue are usually found, macroscopically it is seen as a mottling of the lesion's cut surface. In the acute pneumonia there are three types of pathological changes. Type 1- pulmonary tissue consolidation - is noted in the cranial lobes of lungs and the tissue is dark red, friable and there is no necrosis changes. The type 2 – in a form of the marked consolidation is very often observed in cranial lobes too with red/grey hepatisation, necrosis and suppuration. The extensive consolidation and suppuration are seen during *A. pyogenes* and *F. necrophorum* infections. The 3th type of pathological entity is characteristic for calves that suddenly developed respiratory diseases. In this case there is interstitial emphysema, pulmonary oedema and congestion with alveolar epithelia hyperplasia and hyaline membrane

Viruses are believed to predispose to bacterial infections in two distinct ways. The first, viral agent can cause direct damage to the respiratory clearance mechanism and translocation of bacteria from the upper respiratory tract. The second way, viral infection can interfere with the immune system's ability to respond to bacterial infections. The viruses can affect the leukocytes causing impairment of their function which result in increased susceptibility to infection of *Mannheimia haemolytica* (Jared et al., 2010a). The virus which seen to be the most often responsible for the appearance of BRD is BVD/MD. The virus may suppress the immune system in the affected animals. The infection of BVD/MD leads to inhibition the production of interferon, a decline in the number of leukocytes and the weakening of humoral immunity by reducing production of antibodies it causes bacterial infections

The clinical cases which confirmed the presence of *Mannheimia haemolytica* have a sever character and finishing quick death (Bednarek, 2010). In sick animals are usually found high fever, mucopulurent or pulurent nasal discharge, lacrimation, incidence of painful cough with symptoms of severe shortness of breath, weakness and apathy. In some animals may have been presented watery diarrhea and ill animals not shown willingness to foraging. As a result of this pathogen infection in affected animals occur the extensive damage and inflammation of lung tissue. *M. haemolytica* produces many other potentially virulent factors, among them leukotoxin (Lkt; Hinghlander, 2001; Whitley et al., 1992). The leukotoxin izoform produced by *M. haemolytica* biotype A, serotype 1, has the most visible cytotoxic proprieties in relation to bovine leukocytes. It has been discovered that bovine leukocytes exposed to low doses of exotoxin show reduced phagocytic and killing activity engulfed bacteria. On the other hand, higher concentration of the agent causes complete destruction of the leukocytes leading to their swelling and bursting (Clinkenbeard et al., 1989; Bednarek et al., 2009). The Lkt binds specific leukocyte adhesion molecules, signal-inducing transmembrane pore formation is generated, leading to efflux of K+, influx of Ca2+(Clinkenbeard et al., 1989). Many potentially profitable reactive substances (free radicals, lizosomal enzymes, proteases) in relation to phagocytes (netrophils, monocytes) are realised from the

were detected particularly in relation to: BRSV, BHV-1, BVDV, PI-3 and Adeno-3.

formation (Andrews, 2004).

(Polak, M., 2008).

The initial viral or mycoplasmal diseases are usually mild and are clinically distinguishing. The syndrome can be form subclinical to acute; most are somewhere in between. The initial viral/mycoplasmal diseases causes a moderate fever, sometimes accompanied by constipation. This is followed by rhinitis with a serous-to-mucopulurent discharge and pneumonia with a harsh, hacking cough, tachypnea, dyspnea and diarrhea. The calves very often are depressed, listless and anorectic. The bacterial infection causes intensity these sigs, with higher fevers, more severe dyspnea and depression and sigs of toxaemia. Calves are particularly difficult to auscult and abnormal lungs sounds may be hard to detect. In cases with severe consolidation, the normal breath sounds are replaced by harsh, high-pitched, large airway noises in the anterior-ventral lung fields. When secondary infection with *Pasteurella multocida* occurs the temperature rise to 41 -41.5°C, the area of lung affected is much increased, and increased breath sounds due to congestion are followed by pleuritic friction rub. The acute course is 10 to 14 days. The differential diagnoses should include aspiration pneumonia from improper tubing or feeding practices and purely viral pneumonias such as those caused by IBR, BVD and bovine respiratory syncytial virus (Blood et al., 1983; Smith, 1990).

The laboratory diagnostics of BRD is directly connected with an isolation and identification of suitable species of viruses, bacteria or mycoplasmas presented in a sample tested. These methods are in the various cases the same or similar, but some are specific to a given agent. However, at present the routine diagnostics is divided into two parts. The first one mainly consist of serological methods and the second is nowadays dominated by the molecular biology (PCR and real time-PCR). In the intravital diagnostic process an usual material collected is the nasal swabs or lung lavages and sera samples. On the other hand, postmortem there are collected tissue samples from lungs and parenchymatous organs (liver, kidneys, spleen). The random amplified polymorphic DNA polymerase chain reaction

Bovine Respiratory Syndrome (BRD) Etiopathogenesis, Diagnosis and Control 369

discipline, the fully effective BRD system of treatment should be included three independent steps and the system could be called as "a three-pillar therapeutic strategy of BRD". The first is an elimination of infectious agents using an appropriate antibacterials, the second is modulation of the pulmonary inflammatory reaction and the third – correction of

However, directly before undertaking of the treatment due to the economic considerations and potencial reduction of therapeutic costs, the field cases of the syndrome should be classified into four grades: Grade 1, subclinical disease (therapy is usually not necessary); Grade 2, compensated clinical disease (at this stage, the inflammatory reaction generated tends to limit the impact of the disease on the animal, this clinical form of the disease needs mainly antibacterial therapy); Grade 3, noncompensated clinical disease (at this stage, the inflammatory reaction is excessive and must be controlled by additional use of antiinflammatory drugs); Grade 4, irreversible clinical disease (which threatens the animal's survival, this BRD form is not treated because conceivable profitable effects not compensate

The first element of complex therapeutic strategy of BRD aimed quick pathogenic bacteria elimination, particularly these originated from *Pasteurellacae* family (*M. haemolytica, P. multocida, H. somni*) which as important infectious factors participate in the development of pulmonary lesions and dysfunction associated with the syndrome. These bacteria play a crucial role in the pathologic cascade: therefore, the antibiotic must be administered as soon as possible after the induction of the infection, which is most often clinically characterized by hyperthermia, reduced appetite, and nasal discharge. Antibiotic treatment must be initiated before irreversible damage (characterized by oral breathing, orthopnea, lactatemia, and cyanosis) occurs. Among antibiotics presently used most often are administered long acting antibacterials such as some tetracyklines (oxyteracykline), macrolides (florfenicol, tulathromycin, gamythromycin) and fluoroquinolones (enrofloxacin, marbofloxacin, danofloxacin) with a wider antibacterial spectrum included also mycoplasmas (*M. bovis, M. bovirhinis, Ureaplasma diversum*). Significant role of mycoplasma in BRD etiology is not now questioned, and it is especially important because their effective control is very difficult. It is generally known that the mycoplasmas are resistant to beta-lactames and cephalosporins because of the lack of cell wall. The same resistance can be observed to nalidixic acid, polymyxin, rifamycin, tylosin, lincomycin, tylmicosin, trimethoprim and to sulfonamides (Poumarat *et al*., 1996; Ayling *et al*., 2007). Therefore, in this case there are intensively searching new more effective generations of antibiotics against mycoplasma infection. The most important mycoplasmal etiological agent in BRD i.e. *M. bovis* as other mycoplasmas is sensitive to antibiotics, which inhibit the protein or nucleic acid synthesis. Recently, in cattle respiratory treatment there are recommended antibiotics which were only applied in swine medicine i.e. pleuromutilins (tiamulin, valnemulin). At present it is known too, that tiamulin has also excellent activity against cattle mycoplasmas like *M. bovis*. In addition an analog compound of tiamulin is valnemulin, which has proven to be affective in the control

*M. bovis* infection under field conditions (Stipkovits *et al*., 2001; Tenk, 2005).

Recently, new antibiotic-treatment conceptions of BRD have been presented during the first European Buiatrics Forum in Marseille (2009). There were described three independent conceptions which in shortening forms are called as SISAAB, SILAAB and MILAAB. The

mechanical and secretolitic lung disorders.

costs, and affected animals most often die).

(RAPD-PCR) found excellent correlation between lung and nasal isolates. The nasal passages of sick animals may provide clues as to what strain is present in the lung (Jared et al., 2010b).

In the diagnosis of mycoplasmal infection there are used both microbiological, serological and molecular biology methods. It is worth mentioning that mycoplasmas need specific media (Eaton's or Hayflik's medium) and suitable conditions to grow, i.e. 37ºC and 5% of CO2. Mycoplasma culture methods were also described by Autio et al. (Autio et al., 2007). A characteristic feature of mycoplasmas is their growth on solid media in the form of "fried eggs" (Miles, 1998). Some species of mycoplasmas such as *M. bovis* have the ability to create spot and film reactions and the latest increase their resistance to adverse environmental conditions during culture. In order to diversify the presence of mycoplasmas and bacteria in the material from culture microscope method by Diens's was applied. In this method, mycoplasmal colonies are visible due to their ability to absorb dye (Malinowski & Kłossowska, 2002). Polymerase Chain Reaction (PCR) and its modification - real-time PCR (rt-PCR) are the techniques for identifying mycoplasmas from biological material (Sachse et al. 2010; McAuliffe et al., 2005; Miles et al., 2004; Vasconcellos et al., 2000) but they have some limitations. Technique which is devoid of these limitations is denaturing gradient gel electrophoresis (DGGE) that allows differentiation of sixty seven mycoplasma species from one sample, including thirteen bovine pathogens in this *M. bovis*, *M. dispar*, *M. bovirhinis* and *M. canis* (McAuliffe et al., 2005). However, to detect the presence of anti-mycoplasma antibodies in sera samples there were applied ELISA tests (Ghadersohi, 2005; Bansal et al., 1995).

The diagnosis of bacterial infections involved in BRD is based on many species-specific methods, such as conventional bacterial cultivation (Autio et al. 2007, Angen et al., 1998), phenotyping characterization (Angen et al., 2002), indole reaction (Autio et al., 2007) or molecular biology techniques. From the latest a PCR method was applied for main bacterial agents of the syndrome, such as *P. multocida* (Miflin & Blackall 2001), *M. haemolytica* (Angen et al., 2009) or *H. somni* (Angen et al., 1998).

In order to identify viral infections in the respiratory syndrome there are used methods detecting the presence of both antigens and specific antibodies. An antigen of some virus species, such as BVDV, BHV1, PIV-3 or BRSV can also be identified using isolation test or Elisa methods (Uttenthal et al., 1996; Autio et al., 2007). From molecular biology techniques to identify the viruses species-specific PCR and rt-PCR methods were applied (Autio et al., 2007; Vilcek et al., 1994). However, the presence of specific anti-viral antibodies in sera samples is possible to detect using Elisa techniques (Anderson et al., 2011) and others.
