Lower respiratory dysfunction in neonatal and adult camelids (Proceedings)

A variety of primary lung diseases have been reported in llamas and alpacas in the peer-reviewed literature, although pulmonary dysfunction may be underestimated, due to subtle presenting signs and lack of routine functional analysis of the respiratory track in this species. Diseases such as tuberculosis, nocardiosis, viral pneumonia (coronavirus, adenovirus-associated pneumonia, bovine herpesvirus type 1), pulmonary neoplasia (adenocarcinoma, adenosquamous carcinoma, lymphosarcoma), fungal pneumonia (pulmonary aspergillosis and histoplasmosis), diaphragmatic paralysis, rhodococcus equi-associated lymphadenitis, endogenous lipid pneumonia and cryptococcosis have been identified to date.

The importance of contagious respiratory illness in camelids recently became apparent, when a novel acute respiratory syndrome [ARS] was recognized in California and the East coast of the United States in 2007.1 Clinical signs of disease ranged from mild upper respiratory disease with influenza-like presentation to severe respiratory illness resulting in death. ARS was thus characterized by acute respiratory signs, high fever, and occasional sudden death, which was more common in pregnant alpacas.

A novel coronavirus was subsequently recovered from lung tissue of a clinical case submitted to the California Animal Health and Food Safety Laboratory System (CAHFS). Furthermore, serologic results in 40 animals with a history of ARS and 167 controls, demonstrated a strong association between the presence of novel coronavirus antibodies, exposure to ARS and recent respiratory outbreaks, with an odds ratio of 121 (95% confidence interval: 36.54 and 402.84; P <0.0001).

Autopsy findings in affected animals identified severe pulmonary congestion and edema, often with marked pleural effusion. Histologically, there was severe pulmonary congestion and edema with a marked, diffuse, acute to subacute, interstitial to bronchointerstitial pneumonia. Salient features included free fibrin deposition within the lumen of terminal airways and alveoli, often with hyaline membrane formation. Although pathologic, histologic, and serologic results indicated an relationship between the novel coronavirys and the outbreak of ARS in alpacas, the Koch postulates to prove a true association between the virus and ARS still need to be fullfilled.

The respiratory tract of normal adult camelids

Camelids have 12 ribs, and lamoid lungs are most similar to those of horses. A cardiac notch separates the apical portion of the lung, but there are no lobes, except for a small accessory lobe of the right lung. The mediastinum is complete. The resting respiratory rate of adult llamas or alpacas varies from 10-30 bpm, although normal lung sounds are often muted in heavily fleeced animals.

Standing THORACIC RADIOGRAPHS are generally limited to lateral views in adult camelids. A study of normal thoracic radiographs in llamas reported that pulmonary vessel visualization and definition is similar to that of the normal equine and better than in the bovine. The pulmonary vasculature was generally easily identified in the lung periphery and vessel edge definition was generally good. In only a few instances was peripheral pulmonary vascular visualization judged as poor.

The degree of visualization of the caudal vena cava and descending aorta has been used as an indicator of the presence or absence of pulmonary infiltrates, which may mask vessel margin clarity and/or vessel visualization. The caudal vena cava was always seen in its entirety in the study of normal llamas. The dorsal margin was sharply defined in 15/16 subjects, while the ventral margin was only sharply defined in 7/16 llamas.

The analysis of PLEURAL FLUID may be an important diagnostic tool in certain cases of respiratory illness as recently outlined in alpacas with ARS1 and llamas with pulmonary neoplasia. The preferred site of pleurocentesis is the 6th or 7th intercostal space, 10-15 cm dorsal to the ventrum of the sternum or 2-4 cm dorsal to the costochondral junction of the ribs in normal llamas. Penetration can be made from either side and may utilize a 14-16 gauge needle or cannula (2 inch length) inserted near the cranial border of the rib to avoid intercostal vessels. In normal llamas fluid should flow from the needle at an approximate depth of 2-3cm (1-1.5 inches).

The pleural fluid of 17 clinically healthy adult llamas identified a normal total nucleated cell count of 200–1500 cells/mL with a mean of 576 +/- 361 cells/microliter. In these fluids, small lymphocyte, mononuclear cell, and neutrophil percentages ranged from 80%–100%, 0%–20%, and 0%– 10%, respectively. Pleural fluid total protein concentrations, determined by refractometry, ranged from < 2.5 to 3.5 g/dL. The pleural fluid glucose and lactate concentrations were very similar to the plasma concentrations in normal llamas, with a pleural fluid specific gravity of 1.0133 +/- 0.002, glucose concentration of 135.1 +/- 9.02 mg/dL and lactate of 2.95 +/- 1.34 mg/dL.

The cytologic characteristics of tracheal fluid obtained by TRANSTRACHEAL ASPIRATION have also been described in 17 clinically healthy adult llamas. The technique for transtracheal aspiration was similar to that used in horses. A 10 cm2 area over the ventral mid-cervical region was clipped, aseptically prepared and locally infiltrated with Lidocaine HCl (2% solution).

A stab incision was made through the skin with a #15 scalpel blade to allow insertion of a 10-gauge, through-the-needle Winch catheter between the tracheal rings. Approximately 20 mL of sterile saline was injected through the catheter into the trachea, then immediately aspirated back into the syringe. Cytologic evaluation of TTA fluid revealed that the majority of cells were vacuolated macrophages (60-100%), with 0-40% neutrophils, and fewer lymphocytes (0-2 %), eosinophils (0-3%), and ciliated respiratory epithelial cells (0-10%).

Cytological evaluation of the BRONCHO-ALVEOLAR LAVAGE FLUID (BALF) in healthy alpacas recently identified 58.52 +/-12.36% alveolar macrophages, 30.53 +/-13.78% lymphocytes, 10.95 +/-9.29% neutrophils, 0% mast cells and several ciliated epithelial cells. The percentage of alveolar macrophages and lymphocytes was therefore similar as previously reported in horses, while the mean percentage of neutrophils was higher in alpacas. In this study all animals were anesthetized with a combined intramuscular injection of xylazine (0.5 mg/kg BW), butorphanol (0.05 mg/kg BW) and ketamine (5 mg/kg BW) and maintained in sternal recumbency with the head and neck extended vertically. A protective mouth gag was placed to allow visualization of the larynx with the aid of a laryngoscope.

An enteral feeding tube (Mila International,Inc, Erlanger, KY ) containing a guide wire and measuring 250 cm in length and 6 mm in diameter was inserted into the animal's trachea and advanced until wedged. Following removal of the guide wire, 50 ml of sterile saline was instilled and immediately withdrawn for sampling. Additional 1-3 aliquots were used based on turbidity of the sample obtained. A lateral and a dorso-ventral thoracic radiograph were obtained at the end of the procedure to verify the location of the collection tube. The described BAL technique yielded samples with adequate cellularity and resulted in no complications in healthy alpacas, but it its use may be limited in clinical patients, as short-term, injectable anesthesia was required.

Neonatal lower respiratory disease

There is an apparent paucity of information regarding the clinical manifestation, etiology and outcome of neonatal cria pneumonia. Although pneumonia is a significant cause of morbidity and mortality in neonatal foals, reports are limited to individual case reports in crias. More specifically, pulmonary infection has been associated with gram negative sepsis (Salmonella typhimurium, Escherichia coli, Actinobacillus, and Klebsiella pneumoniae) as well as opportunistic infection of the immune-compromised host. For example, BVDV is known to infect cells that are instrumental in the control of both the innate and acquired immune system. Secondary suppurative bronchopneumonia has thus been diagnosed in BVDV persistently infected crias.

A single report has evaluated the significance of lower respiratory disease in critically ill neonatal camelids to date. Lower airway disease [LAD] was identified in 61/182 (33.5%; 5 llamas, 56 alpacas) critically ill crias less than 4 weeks of age that were admitted to a referral center. A diagnosis of acute respiratory distress syndrome (ARDS) or acute lung injury (ALI) was based on a recent consensus statement11 and could only be obtained in 6/61 (9.8%) crias with LAD.

Of these, four crias survived to discharge. The most commonly identified concurrent diseases in crias with LAD included failure of passive transfer, systemic inflammatory response syndrome, clinical signs of prematurity, neonatal encephalopathy, confirmed sepsis and diarrhea. The latter study further identified specific indicators of LAD in hospitalized patients (manuscript in progress), as well as potential risks of mortality. A total of 142/182 (78%) critically ill crias were discharged alive from the hospital.

Mortality was significantly higher in crias with LAD (39.3%) compared to animals without lower airway dysfunction (13.2%). Similar observations have been previously obtained in neonatal foals at the same cohort. The latter study documented that 35% of neonatal foals with radiographic evidence of pulmonary disease at the time of admission did not survive to discharge, while the mortality of foals without respiratory illness was merely 14%. The majority (54%) of deceased foals were euthanized due to futility of treatment. In contrast, 13/24 (54.2%) crias with LAD died naturally and 9/24 (37.5%) were euthanized. These data suggest that life-threatening respiratory illness may me more insidious in camelids compared to foals.

Open mouth breathing, severe depression, prematurity, recumbency and a diagnosis of systemic inflammatory response syndrome (SIRS) have been significantly associated with the development of lower respiratory disease in neonatal crias. It is important to remember that apparent signs of lung dysfunction may also be associated with non-respiratory conditions such as metabolic derangements (e.g. severe acidosis), pain, abdominal crisis, fever or high environmental temperatures and excitement.

Clinical signs of pulmonary diseases may thus be non-specific and difficult to differentiate by physical examination alone in camelids. We postulate that infectious respiratory diseases in crias are usually part of a multiple-organ, systemic infection or sepsis, similar to observations in foals. Based on a recent report of culture-positive, septic neonatal crias, the most common isolates may include Escherichia coli, Enterococcus spp., Listeria monocytogenes, and Citrobacter spp. Development of respiratory disease in neonates is often related to abnormal perinatal respiratory development, abnormal parturition, aspiration and seeding of primary infectious agents or opportunistic pathogens.

The impact of immune function

Pneumonia is commonly associated with a compromise in the immunologic protection of the neonate. Even in healthy newborns, reduced complement values, defective chemotaxis (directed migration of neutrophils or macrophages) and killing capacity of the neonatal neutrophil contribute to a relatively decreased defense against invading bacteria in other species. Furthermore, cellular immunity and local pulmonary defenses may be reduced in neonatal animals. For example, an immature ciliary apparatus and the presence of fewer alveolar macrophages in neonates in comparison to adult horses lead to decreased bacterial clearance from the lungs.

Failure of passive transfer of immunity (FPT) is a well known risk factor of neonatal infection, including respiratory disease. Affected newborns are not only deprived of specific maternal antibody protection, but their neutrophil function is also seriously impaired.Camelids have a thick layered epitheliochorial placenta, which prevents transplacental transfer of IgG. Pre-colostrum serum IgG levels are therefore low in camelids, with concentrations of 0.26 +/- 10.23 mg/ml. Maximum IgG levels are reached after 24 h of colostrum ingestion. The plateau concentration of 24.52 +/- 8.8 mg IgG/dl subsequently declines after 2-5 weeks. IgG concentrations above 10 mg/ml post partum indicate a successful passive transfer in camelids. Although the neonatal camelid is immunocompetent at birth, it is immunologically naive and therefore dependent on the passively acquired humeral immunity.

Immune responses in llamas and alpacas are unique in that camelid IgG antibodies differ from all other known immunoglobulins. Camelids produce both conventional heterotetrameric IgG (containing paired heavy and light polypeptide chains) common to all vertebrates, as well as functional homodimeric heavy-chain antibodies that lack the light chains. At present, three subclasses of camelid IgG have been identified (IgG1: conventional antibodies; IgG2; IgG3), of which IgG2 and IgG3 do not have light chains. IgG2 and IgG3 consist of dimers of short heavy chains that total approximately 75% of serum IgG. It has been suggested that these unconventional antibodies could represent an evolutionary advantage, being more efficient than conventional immunoglobulins to inhibit microbial enzymes, and thus exerting a more protective immune response against pathogens. Whether these mechanisms enhance protection against neonatal respiratory infection, however, remains speculative.

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