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

Dariusz Bednarek, Monika Szymańska-Czerwińska and Katarzyna Dudek  *National Veterinary Research Institute, Department of Cattle and Sheep Diseases, Poland* 

### **1. Introduction**

362 A Bird's-Eye View of Veterinary Medicine

Walton JS, Halbert GW, Robinson NA & eslie KE. Effects of progesterone and human

Wang JY, Owen FG & Larson LL. Effect of beta-carotene supplementation on reproductive performance of lactating Holstein cows. *J.Dairy Sci*. 1988;71:181-186. Zemjanis R. "Repeat-breeding" or conception failure in cattle. In: *Current Therapy in Theriogenology*. Ed. D.A.Morrow, Philadelphia, WB Saunders, 1980:205-213. Zemjanis, R. The problem of repeat breeding in cattle, *New England Vet. Meeting*, 1963.

breeder dairy cows. *Can J Vet Res* 1990;54:305-308

chorionic gonadotrophin administration five days potsinsemination on plasma and milk concentrations of progesterone and pregnancy rates of normal and repeat

#### **1.1 Etiopathogenesis**

Bovine respiratory disease (BRD) complex is a major disease, classically in the indoor calves and feedlot young cattle. The etiopathogenesis of BRD is multifactorial and complex. In this complex etiology an equivalent role plays both the infectious agents as well as the environmental factors which are called also as environmental stressors (inappropriate livestock management like mistakes in animal nutrition, transport, handling, veterinary interventions etc.) (tab. 1). The most significant pathogens which are involved in the etiopathogenesis of BRD, i.e. suitable species of viruses such as (bovine respiratory syncytial virus (BRSV), parainfluenza virus type 3 (PI3V), bovine herpes virus type 1 (BHV1), bovine viral diarrhea virus (BVDV), are usually associated with concurrent bacterial infections represented by: *Mannheimia haemolytica*, *Pasteurella multocida, Histophilus somni* and others (tab. 1) (Jared et al., 2010a, Kita et al., 1994, Klimentowski et al., 1995) and also mycoplasmal factors such as: *Mycoplasma bovis*, *Mycoplasma bovirhinis*, *Mycoplasma dispar*, *Ureaplasma diversum* and even *Mycoplasma canis* (Szymańska et al., 2010). Among bacteria *M. haemolytica* and *P. multocida* have been traditionally considered as the most common bacterial infectious factors in the BRD etiology (Fulton et al.; Reggiardo et al., 2005). On the other hand the one of the most often isolated mycoplasmal factors from BRD cases is *Mycoplasma bovis*. Its adaptative ability to a host organism increases owing to different versions of the same *vsp* gene family which encode particular adhesive factors of the mycoplasma, i.e. variable surface proteins (Vsps; Razin et al., 1998). The variability of *vsp* gene based on DNA transposition (Lysnyansky, 1996). Therefore the high Vsp variation determines the phenotypic variability of the antigen (Rosengarten et al., 1994) which further increases the virulence of the pathogen. *M. bovis* possess the ability to immunomodulate host defence against infection (Razin et al., 1998), for example inducing a synthesis of proinflammatory agents, i.e. TNF-α and nitric oxide (Jungi, et al., 1996) or stimulation of some acute phase protein production, such as haptoglobulin and serum amyloid A, which represent one of the most important components of acute phase response for cattle (Dudek, 2010). It is known that *M. bovis* acts as a stimulant on major subsets of T-lymphocytes (Vanden Bush, 2003). In contrast, some data show inhibitory properties of the pathogen which come down to its suppressive effect on lymphocytes (Vanden Bush & Rosenbusch, 2002; Thomas et al., 1990)

Bovine Respiratory Syndrome (BRD) Etiopathogenesis, Diagnosis and Control 365

Additionally, main viruses of bovine respiratory system were concomitant factors of the infections. However, the highest correlation between the prevalence and clinical respiratory signs of disease were observed for *U. diversum* when compared with *M. dispar* and *M.* 

In the chronic form of the respiratory syndrome should be also mention about chlamydia, particularly *Chlamydophila psittaci* and *Ch. pecorum* apart from that anaerobes bacteria such as *Clostridium* sp. are often engaged in the etiopathogenesis of the disease. Moreover, the different endogenous factors play also an important etiological role in the BRD etiology. First of all young cattle before one - year - old have an insufficient developed respiratory system for an efficient gas exchange and fully effective and constructive mucociliary clearance mechanisms. Calves have almost by a half lower of the lung surface for gas exchange and higher primary activity of pulmonary ventilation, which in connection with the increased of metabolic demand in these animals is the main cause of low supply of oxygen in the body. The low partial pressure of oxygen causes a significant weakening of the movement of cilia snapshot, phagocytic activity of pulmonary macrophages and a reduction in lung clearance of harmful microorganisms. When an affectivity of pulmonary clearance and local immune response of the lungs are reduced, a variety of infectious agents (viruses, bacteria, mycoplasmas) have better access to lower parts of the respiratory tract

and they can initiate the development of pathological lesions (Lekeux et al., 1995).

While the different pathogens involved in etiology of BRD are presented in the heard throughout the years, the outbreak of the disease and an appearance of clinical symptoms are seasonal. Usually, there are two seasonal peaks of enzootic pneumonia occurred: the first was between October and December and the second from February to May (Andrews, 2004). The sigs of the disease depend on its clinic form i.e. chronic or acute. The chronic form of BRD generally shows no signs. The calves are bright, eats well but may have a slight mucoid or mucopurulent oculonasal discharge, the body temperature is normal or slightly raised at 38.5-39.5°C and dry, explosive cough usually is produced singly. During the acute case of the disease inappetance, pyrexia (40-42°C), dull sweaty coat, mucoid and mucopurulent oculo-nasal discharge, tachypnoea (respiratory rate- RR over 40 breaths per minute) are presented. Moreover, there is a tendency to a persistent coughing. The cough can be harsh, dry, hacking type but sometimes is moist. During the auscultation of the thorax under both inspiration or expiration there are loud, harsh sounds or whistling,

At present, BRD is a great economic problem in cattle husbandry, especially in young and feedlot animals all over the world. It accounts for approximately 30% of the total cattle deaths worldwide and is associated with an annual economic losses of over one billion dollars in North America alone (Adamu, 2007). However, the most often outbreak of acute disease occurs when there are calves from different sources in herds. The congestion and inadequate ventilation are the predisposing factors for the development of BRD. Despite that the different pathogens cause of BRD its initial clinical symptoms are very similar i.e. bronchitis and bronchiolititis. In the first phase of the disease the mucus production in diseased calves increases and its stand over in respiratory tract. This is cause a main cause for handicap of defence mechanism in the course of the respiratory disease and the spread of bacterial infections in lungs, which resulted in the intensification of the primary viral infections. The studies recently conducted in Poland have been found the mixed viral

*bovirhinis* (Autio et al., 2007).

wheezing or squeaking (Andrews, 2004).

and some neutrophil functions (Thomas, et al. 1991). The inhibitory effect of *M. bovis* on the mononuclear cells is not connected with arginine depletion or cytotoxic activity (Thomas et al., 1990). Recently, *M. bovis* is considered as primary pathogen in BRD, not only acting in coinfection conditions. The significance of *M. bovis* in etiopathogenesis of the syndrome is also important with regard to synergistic effect with some infectious factors involved in BRD. For example, *M. bovis* exhibits the colonising synergism with *Mannheimia haemolytica* (Houghton & Gourlay, 1983), whereas coinfection with bovine viral diarrhea virus (BVDV) may increases a pathogenicity of concomitant infectious agents mainly considering suppression of host immune response (Potgieter, 1997; Haines et al., 2001). The disease course during infection with *M. bovis* is generally acute and relates to young cattle (Wikse, 1985). Clinical symptoms mainly limited to dysfunction of the respiratory tract of animals, including cases of fatal diseases, and their intensity decreases with the development of chronic phase of infection (Nicholas & Ayling, 2003). However, complicated infections with *M. bovis* have usually severe course and may take the form of necrosuppurative bronchopneumonia. Pathological changes of the disease are characterized by consolidation areas with nodular lesions containing necrosuppurative material surrounded with fibrous capsule, whereas in histology they are rich in foci infiltrated by degenerating leukocytic cells (Radaelli et al., 2008).


Table 1. The most important etiological causes of BRD

In the complex etiology of BRD also other mycoplasmas, such as *M. dispar*, *M. bovirhinis* or *U. diversum* can play an important role (Autio et al., 2007). These mycoplasma species were concurrently present in 50% of examined herds and bacterial agents of the syndrome, i.e. *P. multocida*, *Arcanobacterium pyogenes* or *M. haemolytica* coexisted with these cases.

and some neutrophil functions (Thomas, et al. 1991). The inhibitory effect of *M. bovis* on the mononuclear cells is not connected with arginine depletion or cytotoxic activity (Thomas et al., 1990). Recently, *M. bovis* is considered as primary pathogen in BRD, not only acting in coinfection conditions. The significance of *M. bovis* in etiopathogenesis of the syndrome is also important with regard to synergistic effect with some infectious factors involved in BRD. For example, *M. bovis* exhibits the colonising synergism with *Mannheimia haemolytica* (Houghton & Gourlay, 1983), whereas coinfection with bovine viral diarrhea virus (BVDV) may increases a pathogenicity of concomitant infectious agents mainly considering suppression of host immune response (Potgieter, 1997; Haines et al., 2001). The disease course during infection with *M. bovis* is generally acute and relates to young cattle (Wikse, 1985). Clinical symptoms mainly limited to dysfunction of the respiratory tract of animals, including cases of fatal diseases, and their intensity decreases with the development of chronic phase of infection (Nicholas & Ayling, 2003). However, complicated infections with *M. bovis* have usually severe course and may take the form of necrosuppurative bronchopneumonia. Pathological changes of the disease are characterized by consolidation areas with nodular lesions containing necrosuppurative material surrounded with fibrous capsule, whereas in histology they are rich in foci infiltrated by degenerating leukocytic cells

**Stress factors Viruses Bacteria** 

*Bovine respiratory syncytial virus (BRSV) Parainfluenza virus type 3* (PI3) *Bovine herpes virus type 1 (BHV)) Bovine viral diarrhoea virus (BVDV) Adenoviruses Rhinovirus Enteroviruses Bovine respiratory coronavirus* 

In the complex etiology of BRD also other mycoplasmas, such as *M. dispar*, *M. bovirhinis* or *U. diversum* can play an important role (Autio et al., 2007). These mycoplasma species were concurrently present in 50% of examined herds and bacterial agents of the syndrome, i.e. *P. multocida*, *Arcanobacterium pyogenes* or *M. haemolytica* coexisted with these cases.

*Mannheimia haemolytica P. multocida Histophilus somni Arcanobacterium pyogenes Streptococcus pneumonie Staphylococcus aureus Chlamydiales spp. Fusobacterium necrophorum Corynebacterium bovis Streptococcus spp. Micrococcus spp.* 

> Mycoplasmas *M. bovis M. bovirhinis M. dipsar M. alkalesscens M. canis M. bovigenitalium Ureaplasma spp. Ureaplasma diversum*

(Radaelli et al., 2008).

Heat Cold Dust Dampness Injury Fatigue Dehydration Hunger Anaxiety Irritant gases Nutrional deficiencies Surgery

Table 1. The most important etiological causes of BRD

Additionally, main viruses of bovine respiratory system were concomitant factors of the infections. However, the highest correlation between the prevalence and clinical respiratory signs of disease were observed for *U. diversum* when compared with *M. dispar* and *M. bovirhinis* (Autio et al., 2007).

In the chronic form of the respiratory syndrome should be also mention about chlamydia, particularly *Chlamydophila psittaci* and *Ch. pecorum* apart from that anaerobes bacteria such as *Clostridium* sp. are often engaged in the etiopathogenesis of the disease. Moreover, the different endogenous factors play also an important etiological role in the BRD etiology. First of all young cattle before one - year - old have an insufficient developed respiratory system for an efficient gas exchange and fully effective and constructive mucociliary clearance mechanisms. Calves have almost by a half lower of the lung surface for gas exchange and higher primary activity of pulmonary ventilation, which in connection with the increased of metabolic demand in these animals is the main cause of low supply of oxygen in the body. The low partial pressure of oxygen causes a significant weakening of the movement of cilia snapshot, phagocytic activity of pulmonary macrophages and a reduction in lung clearance of harmful microorganisms. When an affectivity of pulmonary clearance and local immune response of the lungs are reduced, a variety of infectious agents (viruses, bacteria, mycoplasmas) have better access to lower parts of the respiratory tract and they can initiate the development of pathological lesions (Lekeux et al., 1995).

While the different pathogens involved in etiology of BRD are presented in the heard throughout the years, the outbreak of the disease and an appearance of clinical symptoms are seasonal. Usually, there are two seasonal peaks of enzootic pneumonia occurred: the first was between October and December and the second from February to May (Andrews, 2004). The sigs of the disease depend on its clinic form i.e. chronic or acute. The chronic form of BRD generally shows no signs. The calves are bright, eats well but may have a slight mucoid or mucopurulent oculonasal discharge, the body temperature is normal or slightly raised at 38.5-39.5°C and dry, explosive cough usually is produced singly. During the acute case of the disease inappetance, pyrexia (40-42°C), dull sweaty coat, mucoid and mucopurulent oculo-nasal discharge, tachypnoea (respiratory rate- RR over 40 breaths per minute) are presented. Moreover, there is a tendency to a persistent coughing. The cough can be harsh, dry, hacking type but sometimes is moist. During the auscultation of the thorax under both inspiration or expiration there are loud, harsh sounds or whistling, wheezing or squeaking (Andrews, 2004).

At present, BRD is a great economic problem in cattle husbandry, especially in young and feedlot animals all over the world. It accounts for approximately 30% of the total cattle deaths worldwide and is associated with an annual economic losses of over one billion dollars in North America alone (Adamu, 2007). However, the most often outbreak of acute disease occurs when there are calves from different sources in herds. The congestion and inadequate ventilation are the predisposing factors for the development of BRD. Despite that the different pathogens cause of BRD its initial clinical symptoms are very similar i.e. bronchitis and bronchiolititis. In the first phase of the disease the mucus production in diseased calves increases and its stand over in respiratory tract. This is cause a main cause for handicap of defence mechanism in the course of the respiratory disease and the spread of bacterial infections in lungs, which resulted in the intensification of the primary viral infections. The studies recently conducted in Poland have been found the mixed viral

Bovine Respiratory Syndrome (BRD) Etiopathogenesis, Diagnosis and Control 367

destroy cells and then they stimulate different pathological lesions in host affected lung tissue. The process manifested with an acute lobar fibrionecrotising pneumonia. This histopathological picture is characteristic for acute BRD. All the changes are consequences of Lkt action and the development of lung inflammatory cascade regulated additionaly by some pro-inflamatory cytokines (Bednarek et al., 2009). The clinical cases in which the *M. haemolityca* is included have severe clinical courses. According to the literature the intravenous administration of *M. haemolytica* A1 leukotoxin to clinically healthy calves caused the occurrence of the leukopenia manifested with the significant decrease of total peripheral WBC count with lower values of polymorphonuclear leukocyte and MID cell percentages, the last is a total value of all blood peripheral monoctes, eosinophils and basophils. Moreover, there were significant changes regarding some lymphocyte subpopulations such as: CD2+ (T lymphocytes), CD4+ (T helper lymphocytes) and CD8+ (supresor/cytotoxic lymphocytes). The leukopenia resulted from the toxic influence of *M. haemolytica* A1 Lkt, demonstrating a species-specyfic depletion effect with respect to bovine leukocytes (Bednarek et al., 2008). Additionally, the concentrations of some acute phase proteins (CRP, Cp, Tf, Hp, SAA and also eicosanoids (PGE2, PGF2α, LTB4) had also significantly changes, because there were their higher values after administration of

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

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

leukotoxin (Bednarek et al., 2009, 2010).

(Blood et al., 1983; Smith, 1990).

**2. Diagnosis** 

infections in field outbreaks of BRD (Kita et al., 1994). The highest specific antibody titres were detected particularly in relation to: BRSV, BHV-1, BVDV, PI-3 and Adeno-3.

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 formation (Andrews, 2004).

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 (Polak, M., 2008).

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 destroy cells and then they stimulate different pathological lesions in host affected lung tissue. The process manifested with an acute lobar fibrionecrotising pneumonia. This histopathological picture is characteristic for acute BRD. All the changes are consequences of Lkt action and the development of lung inflammatory cascade regulated additionaly by some pro-inflamatory cytokines (Bednarek et al., 2009). The clinical cases in which the *M. haemolityca* is included have severe clinical courses. According to the literature the intravenous administration of *M. haemolytica* A1 leukotoxin to clinically healthy calves caused the occurrence of the leukopenia manifested with the significant decrease of total peripheral WBC count with lower values of polymorphonuclear leukocyte and MID cell percentages, the last is a total value of all blood peripheral monoctes, eosinophils and basophils. Moreover, there were significant changes regarding some lymphocyte subpopulations such as: CD2+ (T lymphocytes), CD4+ (T helper lymphocytes) and CD8+ (supresor/cytotoxic lymphocytes). The leukopenia resulted from the toxic influence of *M. haemolytica* A1 Lkt, demonstrating a species-specyfic depletion effect with respect to bovine leukocytes (Bednarek et al., 2008). Additionally, the concentrations of some acute phase proteins (CRP, Cp, Tf, Hp, SAA and also eicosanoids (PGE2, PGF2α, LTB4) had also significantly changes, because there were their higher values after administration of leukotoxin (Bednarek et al., 2009, 2010).
