Bovine Respiratory Disease (BRD) is a common respiratory disease in domestic cattle. BRD is caused by various microorganisms, including bacteria, viruses, mycoplasmas, and, less frequently, fungi and parasites, which once established in a herd, easily spread from one animal to another ^{3, 7.}

Due to its multifactorial nature, the diagnosis of BRD results from the combination of clinical observations during a disease episode and the results obtained from samples sent to the laboratory, using one or more analytical methods in combination ^2-9. A holistic approach to diagnosis is, therefore, essential for interpreting the results and making decisions. In this regard, it is important to know how to act to obtain a rapid and accurate diagnosis of BRD in the two possible scenarios in which we face this disease, post-mortem during necropsy or ante-mortem in cases of clinical suspicion.

BRD can vary in its clinical presentation and the severity of symptoms from one animal to another, with acute, subacute, or chronic presentations. For this reason, the clinical history of the outbreak will be of great help in correlating laboratory findings with field observations. In the acute presentation, the disease can cause high morbidity and mortality. In this presentation, not excluding non-infectious factors or the effect of the production system (closed or open), Bovine Respiratory Syncytial Virus (BRSV) is usually involved, although Mannheimia haemolytica can also cause subacute episodes. Chronic cases of BRD are characterized by symptoms that persist for 3-5 weeks. The pathogens typically involved are bacteria from the Pasteurellaceae family, such as P. multocida, H. somni, and M. haemolytica, as well as Mycoplasma bovis, and less frequently, pyogenic, opportunistic, and commensal bacteria. As in acute presentations, in this case it is also possible to detect the presence of respiratory pathogens, such as viruses and pathogenic mycoplasmas, co-infecting the same lung through qPCR (Real-Time Polymerase Chain Reaction). Therefore, knowing the chronology of the outbreak will help discern which pathogens were primary, simultaneous, or secondary in the infection and disease process.

The identification of lesions associated with BRD is crucial to guide any veterinary intervention aimed at controlling the outbreak. In general terms, BRD causes bronchopneumonia, characterized by inflammation and consolidation of lung tissue. Lungs can present areas of congested and reddened tissue as a result of inflammation. In severe cases, there may be hemorrhage in the organ's parenchyma. Lungs with consolidated tissue are firm to the touch and heavy due to the accumulation of exudates. When cut open, they exhibit a firm and solid consistency, typically reddish or grayish, lacking the normal spongy texture.

In acute cases of BRD, fibrinous pleuritis is observed, which is inflammation of the visceral pleura with fibrin accumulation, leading to adhesions between the pleura and the thoracic cavity. If the infection is advanced and involves bacteria like Mannheimia haemolytica or Pasteurella multocida, pulmonary abscesses can be found in various stages of encapsulation and development. Regional lymph nodes, such as tracheobronchial and mediastinal lymph nodes, exhibit various alterations, such as increased size, redness, necrosis, or the presence of abscesses. Finally, in acute cases of BRD with a primary involvement of viruses, it is possible to observe nasal and tracheal lesions, such as hyperemia, ulceration, and the accumulation of mucous or purulent secretions in the nasal cavity and trachea.
 

The three types of samples that can be collected on a farm for BRD diagnosis are blood, nasal secretions, and lung and respiratory tract biopsies. Each type of sample provides different information, and its diagnostic value depends on the course of the disease and the herd's health status. In general, blood is used for antibody screening (vaccination, recent infections with non-endemic pathogens, colostrum), indicating the animal's previous contact with the pathogen but not confirming the disease. Nasal secretions and lung samples are used to confirm the presence of the pathogens responsible for BRD, both ante-mortem and post-mortem. In the latter case, a direct causal relationship cannot be established, but all clinical and laboratory results, taken together, guide the diagnosis.
 

Serum obtained from a blood sample is useful for determining the presence and, in some cases, the relative quantity of antibodies against a pathogen. Serological tests are considered confirmatory only in cases of historically negative populations that have become infected. Any positive animal should be evaluated using a confirmatory method.

It is recommended to collect blood samples by venous puncture using the vacuum tube system. Once collected, the sample should be kept at room temperature until a clot forms (2 hours), and then it can be refrigerated to aid complete retraction. Excessive stress on the animal during collection should be avoided, and conventional syringes and tubes that cause hemolysis should not be used. The released hemoglobin can interfere with laboratory assays.
 

Antibodies in serum are routinely detected using the Enzyme-Linked ImmunoSorbent Assay (ELISA). This is a robust method available in most laboratories, and its mass and automated format make it very popular ¹⁰. ELISA is the screening technique of choice for monitoring populations negative for certain infections.

The main limitation of ELISA for BRD diagnosis is associated with the high prevalence of antibodies in animals, whether due to vaccination, infection, or colostrum ⁸. This requires that each ELISA test be compared with a second test (paired tests) after a gap of 3-4 weeks, or serial sampling at defined time intervals (cross-sectional seroprofiles). This way, changes in antibody levels between successive samples can be evaluated. In the case of paired samples, they must be taken from the same animals, analyzed with the same ELISA method (various brands exist), and the differences in levels between the first and second sample must be significant (2-4 orders of magnitude). If the samples come from calves under 3-6 months of age, the presence of colostral antibodies complicates interpretation. Finally, it should be noted that ELISA serology in a commercial, validated, and mass-used format is only available for viruses and not for most bacteria causing BRD.

Since it is a disease confined to the respiratory system, samples to confirm the presence of pathogens causing BRD are limited to the upper or lower respiratory tract (ante-mortem) and the lungs (post-mortem). However, in some cases, the causative agent can induce extrapulmonary lesions that should be evaluated and recorded during necropsy, as is the case with Bovine Viral Diarrhea Virus (BVDV).

In cases of BRD with mortality, taking samples during post-mortem examination is crucial. However, it is also important to consider taking samples from animals that coexisted with the deceased, as BRD spreads rapidly. For this reason, in vivo samples should be taken from 4-5 individuals and, if possible, from all dead animals. These samples are taken from clinically affected animals, even if the symptoms are mild and nonspecific. The sampling is done using sterile dry swabs in tubes, which are inserted firmly into the nostrils to obtain epithelial tissue and the maximum amount of secretion possible, avoiding trauma. Various types of swabs are available for this purpose ¹. However, a recent study shows that, for the qualitative assessment of the nasal flora in cattle, as well as for the recovery of M. haemolytica, conventional swabs were as effective as larger swabs with covers (catheter-type). In general, commercial swabs of 15-20 cm in length are suitable.

Once the sample is collected, its moisture is sufficient to keep the bacteria alive until it reaches the laboratory for culture, identification, and, if necessary, antibiogram testing in case of symptomatic animals. Therefore, it is not necessary to use transport swabs unless the transit time to the laboratory is days. In the case of H. somni, it is crucial to take the sample aseptically and reduce the transport time to a few hours if bacterial culture is expected.

In a scenario of monitoring the circulation of pathogens involved in BRD, with no mortality but with early nonspecific symptoms, qPCR is a valid alternative for detection ⁶. In this case, swabs can be used in the same way as mentioned for bacterial isolation. However, if the transit time is prolonged, desiccation or exposure to direct light partially degrades the genetic material. This constitutes a limitation since qPCR is based on the detection of intact DNA (mainly bacterial and some viral) and RNA (some viral) for successful detection. As an alternative, there are chemically treated paper matrices called FTA cards, which stands for Flinders Technology Associates cards. They inactivate pathogens while fixing and preserving intact nucleic acids for subsequent detection by qPCR. This way, the sample is safe (non-infectious), and nucleic acids can be detected for a longer period without the limitations of preserving untreated samples (swabs, tissues, body fluids, etc.) ¹¹.

If there are animals with severe symptoms suitable for euthanasia or cases of death due to BRD, it is feasible to take samples of lung tissue and respiratory tracts, both for bacteriology (culture or qPCR depending on the pathogen) and for virology (qPCR) and histopathology. This can be done during field necropsy by biopsy or swabbing of the affected lung parenchyma in situ, or it can be referred to the laboratory by sending representative pieces of lung lesion and unaffected tissue from the same animal. This way, the analyst will take the appropriate samples, and the possibility of cross-contamination will be reduced. In general, it is advisable to avoid sampling animals in an advanced state of decomposition as much as possible, as well as sending small organ fragments. FTA cards become a valid alternative for taking samples from carcasses by vigorous and deep swabbing of the affected lung parenchyma and transferring the sample to FTA.

For shipment to the laboratory, samples should be placed in suitable containers, swabs in their original tubes, and tissues in airtight containers with rigid walls. They should not contain any preservative, should not be washed with water, and should be packaged separately if they belong to different animals. Tissues for histopathology should be sent in 10% (v/v) buffered formalin, leaving one-fifth of the container as an air chamber. All samples should be individually identified with information about the tissue of origin and the collection date. Shipping should be done urgently and in an isothermal container with cold packs. Once desiccated (1-3 hours), FTA cards do not require refrigeration for shipment. They are accompanied by a desiccant and can be sent in a mail envelope with the relevant information.
 

The presence of bacteria and mycoplasmas associated with BRD is evidenced by culture and/or qPCR (Real-Time Polymerase Chain Reaction). Viruses are detected through qPCR since it aims to demonstrate their presence. Histological tests such as pathological anatomy, immunohistochemistry, and in situ hybridization are less common but can be complementary to other methods as long as the samples are obtained and preserved under optimal conditions (fresh and fixed in formalin).

Bacterial culture (BC) is a widely used method for BRD diagnosis, as it allows the isolation of bacteria, their identification, and the performance of antibiograms. However, BC takes time (days) to obtain results, and the results depend on the quality of the sample (autolysis, heterolysis). Some BRD bacteria grow after 24 hours of inoculation (M. haemolytica, P. multocida), while others do not grow under standard conditions (they require specific culture media and conditions) or take 3-5 days to grow after successive passes of the original culture (H. somni, Mycoplasma bovis) ⁴. All these factors delay the acquisition of results and the start of treatment. These disadvantages are shared, although in different contexts, with histopathology, which provides detailed information about tissue changes caused by BRD, such as inflammation, necrosis, fibrosis, or other characteristic histological patterns, but it is strongly affected by sample quality and requires time for execution.

The principle of qPCR is comparable to making a "photocopy" of an original document. If qPCR detects molecules of the pathogen's DNA/RNA in the sample, it uses these molecules as a "template" and copies them billions of times until they are detectable. This makes it a rapid, highly sensitive, and specific method. qPCR does not depend on the microorganism being alive (viable) for detection and is less affected by sample quality than BC or histopathology. Additionally, qPCR is performed immediately upon the sample's arrival at the laboratory. A single sample, such as FTA tissue or secretions, allows for the simultaneous detection of multiple pathogens since it can be done in a multiplex format (multiple PCR in one tube) ⁵. qPCR is currently the method of choice for the diagnosis and monitoring of BRD.

Author: Jaime Maldonado, DVM, MSc, ECPHM Diplomate. Senior Manager, Scientific Manager Unit (HIPRA)

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