Approach to Diagnosis and Treatment of Respiratory Disease of Undetermined Cause (Undifferentiated Ruminant Respiratory Disease)

Amelia R. Woolums

Information regarding diagnosis and treatment of individual infectious agents that can cause ruminant respiratory disease was presented earlier. However, it is common for more than one of these agents to be involved when a group of cattle, sheep, or goats has an outbreak of respiratory disease.

■ History Successful treatment of one or more ruminants with respiratory disease will require identification and alteration of the risk factors associated with ocurrence of the disease; this is particularly true when multiple animals are affected in an outbreak or when endemic disease occurs at an unacceptable rate. Evaluation of the problem begins with collection of a thorough history and is followed by examination of affected animals and the environment. The history questions are directed at management practices that predispose to respiratory disease. It is important for the veterinarian to directly observe as many management practices as possible to ensure that what is described is actually occurring. Examination of the environment includes evaluation of animal housing to determine whether poor ventilation, crowded or dirty housing, or temperature extremes are likely increasing the disease susceptibility of animals.

■ Clinical Signs Once a thorough history is collected, several animals involved in the outbreak should be examined. The degree of fever and severity of abnormal lung sounds were correlated with risk for retreatement for respiratory disease in feeedlot cattle¹; thus clinical examination may be important not only for accurate identification of sick cattle but also for improving estimates of outcome for cattle with respiratory disease. Scoring systems have been developed to aid in the assessment of clinically evident respiratory disease in individual dairy calves and in groups of dairy calves.^2,3 These scoring systems are suitable for use by laypersons trained to assess calves periodically, to facilitate timely diagnosis. A limitation is that the scoring systems do not differentiate between upper and lower respiratory tract disease. A veterinarian or trained technician can address this limitation by using transthoracic ultrasound evaluation (see the Thoracic Ultrasonography and Radiography section later) to determine whether lung lesions are present in calves with a clincial score indicating respiratory disease.

Ruminants affected by bronchopneumonia exhibit signs of respiratory tract inflammation and sometimes systemic inflam­matory response. In early stages, animals stand off by themselves and do not readily approach feed. They hold their heads and ears lower than normal, appear depressed, and move more slowly than normal. As disease progresses, respirations become rapid and shallow, there may be frequent licking of the muzzle, and a moist cough is often present. Animals may have a fever of 40° to 41° C (104° to 105.8° F), and as the disease progresses they appear gaunt, have deep labored respirations, and may hold the head extended. Dyspnea may be both inspiratory and expiratory. Ocular and nasal discharges progress from serous to mucopurulent.

Nasal discharge, dyspnea, abnormal lung sounds, cough, and high fevers are cardinal signs of bronchopneumonia. Other respiratory tract conditions that must be considered as dif­ferential diagnoses include interstitial pneumonia, pulmonary edema, pleuritis, laryngitis, tracheitis, and lungworms. Rare conditions include thoracic neoplasia and diaphragmatic hernia. Nonrespiratory diseases that may result in abnormal respiratory signs include septicemia, heart failure, acid-base imbalances, and poisonings such as nitrate toxicity. In sheep and goats, ovine progressive pneumonia (OPP), caprine arthritis and encephalitis (CAE), and lung or mediastinal abscesses caused by C. pseudotuberculosis are additional differential diagnoses.

Because ruminants are often housed in groups and are often not acclimated to individual handling, signs of respiratory disease in one or more individuals are often first noticed from a distance rather than from “hands-on” examination. Early signs of bronchopneumonia include mildly depressed attitude and appetite; thus caretakers may first identify animals with bronchopneumonia when they are offered feed, at which time affected animals fail to come forward to consume feed with normal enthusiasm. A standardized system to improve recogni­tion of sick calves can be used to enhance detection of calves with respiratory disease in group pens.⁴

Groups at particularly high risk for respiratory disease, such as animals that have been recently weaned and/or transported by truck or trailer, should be evaluated no less than once per day for at least the first week or two after weaning or transport to facilitate timely identification of new cases.

■ Diagnostic Evaluation OfUndifferentiated Rumi­nant Respiratory Disease If clinical examination of an individual or group of cattle, sheep, or goats indicates that respiratory disease is present, it is not always necessary to pursue diagnostic testing. Occasional outbreaks of respiratory disease occur in groups of ruminants, and such occasional outbreaks can often be managed successfully with administra­tive symptomatic therapy alone. Similarly, individuals with a first-time clinical diagnosis of bronchopneumonia may respond completely to presumptive therapy in the absence of any diagnosis. However, if recurrent outbreaks occur or if animals do not respond appropriately to symptomatic therapy, then diagnostic tests are warranted to better characterize the nature of the problem and to help determine the reason for treatment failure. In such situations a careful evaluation of management practices and facility design is also warranted to ensure that all practices possible are in place to maximize the ability of animals to resist respiratory infection. Management practices important to minimizing respiratory infection are described in the Prevention and Control of Undifferentiated Ruminant Respiratory Disease section later.

Thoracic ultrasonography and radiography. The use of transthoracic ultrasound to identify evidence of lung consolidation, pleural effusion or fibrin deposition, and other abnormalities consistent with bronchopneumonia is feasible and helpful if an ultrasound machine is available for use by an individual properly trained in ultrasonographic evaluation and if animals can be restrained for assessment. An ultrasound machine used for pregnacy diagnosis, one with a transrectal probe, is well suited for on-farm transthoracic evaluation. Seventy percent isopropyl alcohol can be used as a transducing agent; it is not necessary to clip the hair unless excessive mud is present. On-farm use of ultrasonography has been evaluated for the diagnosis of bronchopneumonia in feedlot cattle and dairy calves. The procedure is feasible in dairy calves, where evaluation of the tenth through first intercostal spaces on the right side of the thorax and the tenth through second intercostal spaces on the left side of the thorax is possible, and with some practice it allows the practitioner to assess all regions where lesions consistent with bronchopneumonia are likely to occur. While transthoracic ultrasonography can be used to identify lesions of bronchopneumnonia in feedlot cattle and other older animals, due to heavy forelimb musculature it is often not possible to evalute the first and second intercostal spaces, thus some disease is more likely to be missed. Chutes that are necessary to restrain beef cattle also make ultrasonography more logistically difficult, as the chute may obstruct sites where the probe must be placed and puts the operator at more risk of injury. The sensitivity of transthoracic ultrasound evaluation for the diagnosis of lung consolidation consistent with bron­chopneumonia in dairy calves has been estimated to range from 66% to 94%, and the specificity ranges from 88% to 100%.^5,6 The reader is referred to a detailed review for more information regarding on-farm use of transthroacic ultraso­nography for the diagnosis of lower respiratory tract disease in calves.⁶

When used in conjunction with clinical scoring systems, transthoracic ultrasonography allows identification and dis­crimination of calves with clinical disease limited to the upper respiratory tract, clinical disease including lung disease, or subclinical lung disease.

Although the value of ultrasonography for the diagnosis of bronchopneumonia in sheep and goats has not been extensively assessed, the technique should also be feasible for use in these species, assuming that the probe can be placed into contact with skin overlying intercostal spaces. This may be difficult for sheep in full fleece but should not be a problem for goats or sheep with little or no wool.

Thoracic radiography is useful for confirmation of pulo- monary changes consistent with bronchopneumonia when suitable radiographic equipment is avaiable. For larger cattle this means that radiography is only possible in referral hospitals. On-farm radiography may be feasible for calves and sheep and goats, by use of portable equipment; however, it does carry the risk of radiation exposure to personnel and is likely only cost-effective for valuable individuals.

■ Clinical Pathology and Assessment of Passive Immunity Complete blood cell counts or serum biochemical analyses are rarely of much value in diagnosis of respiratory disease in ruminants. Some viral infections may cause leuko­penia, but when bacterial pneumonia is superimposed the WBC count may be elevated. Ahimals with bacterial pneumonia may have an inflammatory leukogram characterized by a leukocytosis with a mature neutrophilia, possibly with a left shift; hyperfi­brinogenemia is likely to occur. Animals with chronic pneumonia may have a normal WBC count even when there is significant pulmonary pathology.

Failure of passive transfer is a major risk factor for pneumonia in calves,^8,9 so investigation of respiratory disease outbreaks in calves should include an evaluation of passive transfer status by measurement of immunoglobulins or total solids in the serum of a subset of calves 1 to 7 days of age. Measurement of serum total solids with an optical, digital, or Brix refrac­tometer is a practical, satisfactory procedure for estimation of serum immunoglobulin concentrations in groups of calves.

■ Necropsy Findings The value of necropsy findings to confirm the cause of death, particularly in animals that die unexpectedly, cannot be overemphasized. Much valuable information may be gained before a disease outbreak causes excessive mortality if early cases that die are subjected to timely necropsy. On a herd level, establishing a practice of subjecting all animals that die to at least gross necropsy can aid greatly in maintaining management practices that limit animal disease and death; this is because the actual cause of death identified at necropsy can sometimes be unexpected. For example, if an animal is assumed to have died of pneumonia, but it actually died of acute enterocolitis, then inappropriate control measures may be undertaken.

The cost of having animals sent to the local diagnostic laboratory for full necropsy may dissuade some owners from allowing necropsies. However, useful information can be gained simply by gross necropsy evaluation performed on the farm by the local veterinarian or, on larger operations, by staff members trained by the supervising veterinarian. With some practice veterinarians can develop confidence in identifying the major differentiating gross features of common diseases, and they can thus ensure that management is aimed at control of disease of the correct organ system. Moreover, there are some identifiable characteristics typical of the major infectious causes of bronchopneumonia. Gross necropsy can help the veterinarian make a more accurate list of differential diagnoses for outbreaks of bronchopneumonia, allowing the development of a more appropriate plan for treatment and prevention. Gross pathologic features of disease caused by infectious respiratory agents are described elsewhere in this chapter and are sum­marized briefly below. A review of the pathology of common pulmonary diseases of cattle is available for additional information.¹⁰

A respiratory necropsy should include assessment of the upper airways and trachea. Fibrinopurulent material in the larynx is evident in cattle with necrotic laryngitis. Infection with BHV-1 (infectious bovine rhinotracheitis virus [IBRV]) causes generalized reddening (congestion) and small raised, red or pale plaques on the mucosa of the nasal passages and trachea; more severe cases have dark red, hemorrhagic changes, possibly with yellow-brown exudate adherent to the mucosa (fibrinopurulent tracheitis) (see Color Plate 31.2).

Bronchopneumonia in recently transported cattle (shipping fever pneumonia) is most commonly a fibrinopurulent bron­chopneumonia. The cranioventral areas of affected lungs are dark red to gray-brown in color, firm, and heavy. Bronchial lymph nodes are swollen, wet, and dark red. The inflamed lung and parietal pleura are sometimes covered with variable amounts of yellow fibrin, and the pleural cavity may contain straw-colored fluid. Fibrinous pleuritis usually indicates the presence of M. haemolytica or H. somni, or possibly B. trehalosi. These species can also cause necrosis of lung, which will be firm and brown to gray, and which may contain dark red, wedge-shaped lesions (infarcts) that are caused by thrombosis of an artery supplying the region. Large amounts of straw­colored exudate are often present in the pleural space. Chronic cases have multiple abscesses and pleural adhesions. As much as 60% to 80% of the lung tissue is often involved in fatal cases of severe bacterial pneumonia. In severe cases the dorsal regions of the caudal lobes often are mottled by interspersed patches of inflammation and normal parenchyma. In some cases, forced respirations result in vesicular to bullous pockets of emphysema in the dorsal areas of the caudal lobe (see Color Plate 31.3). These changes can also be seen with primary BRSV infections or with AIP. Focal or multifocal areas of firm white to yellow material that look like abscesses may actually be caseous necrosis caused by M. bovis (see Color Plates 31.6 and 31.7). Abscesses containing caseous or liquid purulent material may also be caused by T. pyogenes or anaerobic bacteria; these findings indicate chronicity.

Bronchopneumonia in housed dairy calves is less often fibrinous, but rather is characterized by the presence of firm, collapsed, dark red lobules in the cranioventral and possibly the caudal ventral lung (Color Plate 31.8); in severe cases, whole lobes of the lung may be affected. Firm, dark red lobules without fibrin on the pleura are common in pneumonia caused by P. multocida, M. bovis, or other mycoplasmas. Infection with BRSV and PIV-3 can also cause lobular consolidation of the ventral lung. M. haemolytica or H. somni may also be isolated from such lesions even in the absence of fibrinous pleuritis.

Animals that die after chronic persistent coughing, dyspnea, and weight loss often exhibit lesions of chronic suppurative pneumonia. Bronchi and bronchioles are filled with purulent exudate, there are multiple mature lung abscesses, and greatly dilated bronchioles contain malodorous exudate. When bron­chiectasis is severe, the lung lobes have a nodular appearance.

Although gross necropsy alone can be very helpful in generating an accurate list of differential diagnoses, histopatho­logic and microbiologic findings may add valuable information, particularly in situations where symptomatic treatment has failed. Gross necropsy findings alone are not always definitive; for example, in one study of AIP in feedlot cattle, only 67% of the cases that were diagnosed with AIP based on clinical and gross pathologic findings were confirmed by histopathol- 11

ogy. Iherefore in outbreaks with relatively high mortality or in cases in which therapy is not yielding expected results, the cost of full necropsy of two or three typical cases is likely to be well worth the expense.

If full necropsy at a local diagnostic laboratory is to be undertaken, two or more animals showing signs that are typical of the early stages of the disease outbreak should be sent for euthanasia and necropsy. Although the producer may be reluctant to euthanize animals that may recover and would rather send animals with chronic, nonresponsive disease, the chronic cases are unlikely to yield information relevant to the primary problem.

Microbiologic Tests

Samples for identification of viral and bacterial pathogens associated with cases of respiratory disease can be collected antemortem from live animals or at necropsy of animals that have died or been euthanized. It is important to remember that administration of modified live viral or live bacterial vaccines, particularly intranasal vaccines, in the 4 to 8 weeks preceding diagnosis may complicate interpretation of test results, as efforts to diagnose wild-type agents presumably contributing to disease may instead identify vaccine strains of virus or bacteria that are likely to be shed for some time after vaccination. Remember also that recent vaccination may induce seroconver­sion and can complicate the use of serologic testing to diagnose the cause of a respiratory disease outbreak.

Appropriate samples to collect from live animals include nasal swabs, guarded nasopharyngeal swabs, TTA, or BAL. Each of these samples has benefits and drawbacks. Nasal swabs are usually collected with a 6-inch (13.5-cm) swab that is not guarded (protected) from contamination. After sampling, the swab is put into transport medium that may come with the swab or may be obtained from a veterinary diagnostic laboratory. Guarded nasopharyngeal swabs are usually collected using a longer swab guarded by an outer sheath, such as is used for mare or cow uterine culture. While these swabs are unwieldy for sampling young calves, they allow the swab to be protected from contamination by commensal bacteria in the outer nasal passage, providing a theoretically more representative sample of viruses and bacteria in the nasopharynx that are more likely contributing to respiratory disease.

Although respiratory viruses can be shed by cattle without signs of respiratory disease, if viruses are identified in animals with respiratory disease they should be considered contributing factors. Identification of bacterial respiratory pathogens such as M. haemolytica, P. multocida, H. somni, or M. bovis from nasal or guarded nasopharyngeal swabs requires some interpetation. Because these bacteria are considered part of the normal flora of the upper respiratory tract, their identification in cattle without signs of respiratory disease is not significant. However, in calves with clinical signs of bronchopneumonia, identification of these agents on nasal or guarded nasopharyngeal swabs has been shown to agree moderately to strongly with the results of culture of materials from the lower respiratory tract, such as TTA, BAL, or materials collected at necropsy.^12-14 Guarded nasopharyngeal swabs are considered by many to be superior for the identification of bacterial pathogens because they are less likely to result in cultures that are overgown by contami­nants. Others have suggested that BAL is superior to guarded nasopharyngeal swabs for identification of bacterial pathogens¹⁵; however, BAL is technically more demanding and time consum­ing. Overall, for sampling multiple individuals in a group of calves with respiratory disease, guarded nasopharyngeal swabs provide a reasonably good representation of the agents causing disease in the respiratory tract. However, for valuable individu­als, TTA or BAL is the preferred method of sampling to identify infectious agents causing bronchopneumonia.

Proper handling and transport of the specimens collected from live animals or at necropsy is critical to maximize the chance of an accurate diagnosis. The veterinarian is encouraged to contact the diagnostic laboratory if he or she is uncertain about the proper methods of sample collection and transport; most laboratories now post this information on their websites, making it easy to find for those with internet access. See Table 31.14 for guidelines for identifying common infectious respira­tory pathogens.

Viruses

A specific viral diagnosis requires laboratory confirmation. Most laboratories direct their diagnostic efforts toward the viruses for which vaccines are available. Diagnosis of other respiratory viruses may require the assistance of specialized laboratories.

VIRUS ISOLATION. Virus isolation is time consuming and expensive, but it can be a sensitive method for identifying some viruses. Virus isolation is performed in cell culture, with subsequent use of PCR, immunofluorescence, or IHC to identify the specific virus isolated. A variety of specimens can be tested, including nasal, nasopharyngeal, conjunctival, and tracheal swabs; TTA; BAL; and a variety of respiratory tract tissues that can be obtained at postmortem examination. Fluids, tissues, and swabs may be frozen; however, freezing may kill some of the virus in the sample. Alternatively, swabs and tissue specimens may be placed in a viral transport medium and kept refrigerated or on ice packs until arrival at the diagnostic laboratory, prefer­ably within 24 hours. In general, better success at virus isolation is obtained when specimens are collected in the acute phase of disease.

DETECTION OF VIRAL ANTIGENS. Immunofluorescent anti­body (IFA) staining is a rapid method for identifying specific respiratory viruses. Antemortem identification can be made in cells in material obtained from nasal, nasopharyngeal, or conjunctival swabs or from cells obtained by TTA or BAL. Postmortem identification can be made from frozen tissue sec­tions prepared from a variety of respiratory tract tissues. IHC can also be used to test cells from swabs or fixed tissue. This is a useful procedure that allows histologic examination of tissues in conjunction with specific identification of the causative agent.

Antigen capture enzyme immunoassay (EIA) provides a rapid means for detecting certain viruses. These tests can be performed on fluids or homogenates of tissues obtained from the respiratory tract. Commercially available antigen capture EIAs are available for diagnosis of HRSV infections in infants and young children; these are also capable of detecting BRSV¹⁶ and are used in some veterinary diagnostic laboratories. The same technique has been developed for the detection of BVDV and is in use for screening serum samples to detect cattle persistently infected with BVDV.

DETECTION OF VIRAL NUCLEIC ACIDS. Testing material from swabs, fluids, or tissues for viral nucleic acid by PCR (for DNA viruses) or RT-PCR (for RNA viruses) is widely available at veterinary diagnostic laboratories. One benefit of using nucleic acid detection to identify pathogens is that the pathogen does not have to be viable to be identified; this can be a particular benefit for relatively labile viruses such as BRSV

SEROLOGIC DIAGNOSIS. Retrospective diagnosis of viral infections can be made by determining antibody titers in paired sera from individual animals. The first sample is collected in the acute phase of the disease (the “acute” sample), and the second is collected 2 to 4 weeks later (the “convalescent” sample). In a respiratory disease outbreak, multiple animals should be tested to achieve a serologic diagnosis; it is typical for seroconversion to be identified in only a subset of animals tested in any outbreak. Serologic diagnosis is made by dem­onstrating at least a fourfold increase (i.e., at least two dilutions) in antibody titer in the convalescent sample as compared with the acute sample; a fourfold fall in titer from the acute to the convalescent sample also indicates recent infection. Because day-to-day variation in results of tests used for serologic diagnosis is typical, the acute and convalescent samples should be run by the laboratory on the same day. Thus the acute serum samples should be processed and stored in the freezer by the local veterinarian and later shipped together with the convalescent samples. Because infections in young ruminants can occur in the presence of passively derived antibodies, seroconversion may not always occur during outbreaks of bronchopneumonia involving young animals.

A variety of serologic tests are available for antibody determinations, but most laboratories use a serum neutralization (SN) test (also known as a virus neutralization [VN] test) for identification of antibodies to viruses. It is important to remember that SN tests take several days to run. Some labo­ratories use more rapid procedures such as ELISA or IFA for determination of virus-specific serum antibody titers. A hemagglutination-inhibition test can also be used for PIV-3, influenza D virus, and respiratory coronavirus.

Bacteria

A variety of bacteria have been isolated by aerobic culture of materials collected from the respiratory tract of ruminants with respiratory disease. However, the most frequent and important isolates are M. haemolytica, P. multocida, H. somni, and M. bovis. Other mycoplasmas such as M. dispar or M. ovipneumoniae may be important in some cases. The isolation of T. pyogenes, coliforms, or anaerobic bacteria often is indicative of chronic pneumonia or aspiration pneumonia and may be associated with lung abscessation.

Before bacterial culture is attempted, the status of any recent antibacterial therapy should be determined. If possible, speci­mens from untreated cattle should be collected. It is important to remember that M. haemolytica, P. multocida, H. somni, and mycoplasmas are normal inhabitants of the nasal passages of cattle and may be cultured from respiratory tissue in the absence of respiratory disease. Special culture methods are required to identify H. somni and mycoplasmas, and many diagnostic labs will not test for these organisms unless testing is specifically requested. Moreover, most laboratories will not identify the species of mycoplasmas cultured unless that is requested; thus an isolate may be identified only as “Mycoplasma spp.” If involvement with M. bovis is to be confirmed, the veterinarian will likely need to specifically request species identification for any mycoplasmas isolated. Specific identification of M. bovis is most often done by IFA, IHC, or PCR. Some mycoplasmas are more hardy or easier to grow than others; for example, it is not unusual for M. bovis to overgrow M. dispar when both are present in the same sample. Culture of mycoplasmas typically takes at least 1 week, and laboratories often call a sample negative only after it has been subcultured at least once, which usually requires at least an additional week. Thus it takes longer to get the final results of mycoplasma testing than testing for other bacteria.

When sampling lung tissue for bacterial infection at necropsy, contamination of the sample with environmental agents can best be prevented by first searing the lung surface with a heated spatula or knife, then making an incision with a sterile scalpel, followed by sampling with a swab through the incision. However, it may not be practical to sear the surface of the lung in the field setting. Alternatively, sections of lung and other tissue samples can be sent in sterile, sealed containers on ice to the diagnostic laboratory for appropriate sampling for bacteria.

DETECTION OF BACTERIAL ANTIGENS OR NUCLEIC ACIDS. It is common for diagnostic laboratories to use IHC for identifica­tion of bacteria in formalin-fixed tissues. This technique offers many advantages, including correlation of the pathogenic organism with the lesion and detection of pathogens not found on bacterial culture because of overgrowth of other organisms such as T. pyogenes or P. multocida. The use of PCR for identifica­tion of bacterial agents in a variety of samples is also increasingly available, and this may be more sensitive than culture.¹⁷ The veterinarian is encouraged to contact the diagnostic bacteriolo­gist at the local diagnostic laboratory or check the laboratory website for information regarding which tests are available. MALDI-TOFMS is used by many diagnostic laboratories for rapid identification of bacteria once they have been isolated by culture.

SEROLOGIC TESTING FOR BACTERIAL PATHOGENS. Diag­nosis of infection with bacterial pathogens can be attempted retrospectively through the use of serologic testing as described earlier for viral pathogens. However, the use of serologic tests for identification of infection by bacterial respiratory pathogens has mostly been limited to use in research. Thus these tests are not likely to be widely available in veterinary diagnostic laboratories.

OTHER APPROACHES TO DIAGNOSIS OF RESPIRATORY DISEASE. The use of a computer-aided electronic stethoscope has been shown to have relatively good sensitivity and specificity, compared to pen checking, for identification of respiratory disease in feedlot cattle.¹⁸

Remote assessment of feeding and drinking behavior, infrared thermography, and remote detection of fever by rumen boluses or ear tags have all been used in research settings to identify cattle that have signs of illness typical of BRD. At present these approaches are not yet practical, reliable, and cost-effective for the routine diagnosis of respiratory disease. However, they will likely be the focus of continued research, and one or more of these methods may eventually become applicable for routine use in the future.