The ferret spleen is a crescent-shaped, brown to maroon organ located in the left cranial quadrant of the abdomen, caudal to the liver and along the greater curvature of the stomach, attached by the gastrosplenic ligament (Figure 1A). Remove the spleen by severing the gastrosplenic ligament, collect small pieces from different regions, and pool for virus titration. Additional segments of the spleen may be homogenized to purify splenocytes; specimens can also be collected in formalin for histology.⁵⁰

Viruses exhibiting high virulence in ferrets are frequently recovered from the spleen.^45,52 Virus-specific changes are not commonly noted in the spleen, but splenomegaly or increased cellularity from splenic suspensions has been noted after influenza virus infection.⁴⁹ Viral antigen detection and significant cytokine and chemokine responses have been reported in the spleen after infection with highly virulent influenza viruses.⁴⁸ Given the importance of the spleen in both humoral and cell-mediated immune responses, isolated splenocytes from virus-infected ferrets can be used to assess cellular immune responses either immediately or for subsequent stimulation ex vivo to evaluate cross-reactive immune responses.^49,50

The kidneys are retroperitoneal in the sublumbar region of the abdominal cavity on either side of the vertebral column, dorsally apposed to the subaxial muscles and ventrally enveloped by a thin, variably fatty peritoneum (Figure 1A). Small pieces are cut from each kidney and pooled for virus titration. Similar to the spleen, infectious virus and modulation of proinflammatory cytokines and chemokines are typically only detected in the kidneys after inoculation with highly virulent strains of influenza virus.^48,52 Specific virus-related gross pathology is not generally observed. Despite the sporadic nature of influenza virus detection in kidney tissue, gross and histopathologic examination of kidneys can be conducted to evaluate for other underlying renal disease that can contribute to disease progression. Furthermore, this information may be useful in interpreting clinical blood chemistry panels that include indicators of kidney function.⁴³ Adrenal glands located at the craniomedial poles of the kidneys may also be sampled; viral titers have been reported in this tissue.⁷⁰

Influenza virus may be sporadically detected in gastrointestinal samples (either in tissue specimens or rectal swabs), so intestinal tissue is routinely collected. The ferret alimentary tract consists of the esophagus, a simple monogastric stomach, the small intestines (including duodenum), jejunoileum, and the large intestines (Figure 1A). Small pieces of tissue that are free of digestive matter from the duodenum, jejunoileum, and descending colon (proximal to the rectum) are most frequently sampled; tissues may be pooled for a representative intestinal sample or tested individually.^71,72 Avoid scraping the mucosa when removing digesta to prevent loss of tissue during collection; alternatively, rinsing the specimen with sterile saline can be used to remove digesta. Noting the presence or absence of food in the stomach can inform assessments of anorexia and weight loss after infection, although stomach tissue is not frequently collected.

Selected viruses that have been associated with gastrointestinal distress and vomiting in humans have been recovered at higher rates from intestinal tissue of infected ferrets⁷²; however, gross pathology (observation of necrotic tissue, hematomas, or hemorrhages in tissue or mesentery) is typically only present in animals infected with viruses exhibiting high virulence in ferrets. Histopathologic and immunohistochemical examination is more commonly performed on tissue from the jejunoileum, but necrosis and viral antigen have been reported in both small and large intestinal tissue from virus-infected ferrets.^71,73,74 Virus has also been detected in the mesenteric lymph nodes after both intranasal and intragastric virus inoculation; changes in leukocyte subsets have been reported in this tissue.^49,73 Although the esophagus is not frequently collected, standard intranasal inoculation may lead to deposition of influenza virus in the esophagus because of swallowing of inoculum, and infectious virus has been detected in this tissue.^75,76

The pancreas is an elongated, pale tan, glandular organ, with the body of the pancreas located closely adjacent to the pylorus and extension of the left and right limbs toward the stomach and duodenum, respectively (Figure 1B). Although not routinely sampled, necrosis of the pancreatic duct epithelium with concurrent detection of viral antigen has been reported after infection with highly virulent H5N1 viruses, with changes in viral titers and cytokine expression.^51,52,57,71

The ferret liver, located caudal to the diaphragm and cranial to the stomach (Figure 1A), consists of six lobes (left and right lateral, left and right medial, quadrate, and caudate) and is uniformly dark red in appearance. Discoloration or petechiation in one or multiple lobes may be observed. Small pieces of liver are collected from multiple lobes and pooled as a representative sample for titration. If necessary, entire lobes may be removed if pathology is observed, by loafing and placing sections in fixative for subsequent histopathologic analysis, which may be useful in interpreting clinical blood chemistry panels that include indicators of liver function.⁷⁷ Viral titers in the liver are generally low among highly virulent influenza viruses^45,78 and may or may not be associated with viremia, but have been reported after multiple inoculation routes.^71,73 Viral antigen has been detected in hepatocytes, and altered cytokine expression has been reported in liver samples from influenza virus–infected ferrets.^48,52,57

The gallbladder is a single-lobed, brownish green sac located between the quadrate and right medial lobes of the liver. The bile duct opens into the lumen of the duodenum via a major duodenal papilla, along with the pancreatic duct. Although not commonly sampled after influenza virus infection, bile duct necrosis and detection of viral antigen in bile duct epithelia have been reported after infection with virulent influenza viruses.⁷¹ Visual examination for lesions at necropsy is prudent, as ferrets exhibiting severe disease may possess discolored and/or distended gallbladders in conjunction with severe, multiorgan involvement. Testing the patency of the common bile duct is performed by manually expressing the gallbladder and monitoring excretion into the duodenal lumen, but care must be taken not to allow bile, which is highly caustic, to contaminate other tissues that have not yet been sampled.

Collection and titration of tracheal tissue may provide useful information regarding the scope of virus replication throughout the ferret respiratory tract, especially when interpreting viral load in nasal wash specimens, which can be inclusive of virus released from multiple sites beyond the nasal passage.¹⁹ Tracheal samples collected just distal to the larynx (cranial region) may possess differential distributions of sialic acid moieties and levels of infectious virus compared with samples collected proximal to the mainstem bronchi (caudal region).^3,19 As such, uniformity in collection method between animals within a group, or between different groups in a study, is essential.

Removal of the ribcage by transecting the ribs at the costochondral junctions bilaterally and cutting away the pleural attachments will reveal the thoracic cavity (Figure 1B). The trachea may be sampled before or after removal of the lower respiratory tract en bloc by bisecting at the mid to distal trachea; care should be taken to avoid cross contamination of this tissue. Pieces of the trachea (1 to 2 cm) are collected using scissors for virus titration, and may be pooled or kept separate. Avoid the esophagus during manipulation of the trachea, and remove visible connective tissue attached to the surface of the trachea (Figure 1B). Alternatively, the trachea may be removed as part of the pluck. The pluck consists of the tongue, tonsils, larynx, thyroids, trachea, lungs, and heart removed en bloc; with the ribcage removed, this is achieved via a ventral approach through the mandible to dissect out the tongue and subsequent detachment of the entire tongue-to-lung segment as one unit, bearing in mind that hyoid bones may need to be transected with rongeurs to release the tissue cranially.

It is beneficial to leave a short section of the trachea attached to the bronchi to facilitate orientation of this tissue for histopathologic purposes and for intra-alveolar perfusion of lung tissue with formalin once all samples from respiratory tissues have been collected. Histologically, mild to moderate tracheitis has been reported.⁷⁹ Formalin-fixed tracheal tissue can also be used for subsequent immunohistochemistry applications.⁵² Elevated levels of proinflammatory cytokines and chemokines have been reported in tracheal tissue after in vivo ferret infection or after ex vivo infection of differentiated ferret primary tracheal epithelial cells.^53,54 Infectious virus has also been recovered from tracheobronchial lymph nodes,⁷³ which can be found adjacent to the trachea and near its bifurcation into the mainstem bronchi.

Viral titers have been infrequently reported in the heart for select H7N9 and H5N1 viruses, also associated with viremia. Immunohistochemical assays of heart tissue have been negative, with the exception of rare reports of viral antigen detection.^47,57,78,79 One to two small pieces of myocardium are typically sufficient for virus titration. Avoid cross contamination of this tissue with neighboring lung tissue, which may contain higher viral loads (Figure 1B). Proinflammatory cytokines and chemokines have been reported in heart tissue infected by influenza viruses; gross pathology is not typically observed in the ferret heart.^48,52

Just as collection and titration of nasal wash specimens may inform and support data collected from other upper respiratory tract tissues sampled during necropsy, collection and titration of wash specimens from the lower respiratory tract may be similarly beneficial. Bronchoalveolar lavage fluid may be collected longitudinally from anesthetized ferrets, by using an endotracheal tube to retrieve aspirate,⁶⁰ but is most commonly collected postmortem by flushing the trachea and lungs with sterile fluid and collecting the aspirate.⁵⁸ Bronchoalveolar lavage fluid may be titered for the presence of infectious virus and is often concurrently evaluated to measure cellular immune responses or tested for the presence of antibodies by enzyme-linked immunosorbent assay.^49,58–60 Ferret alveolar macrophages, collected by this method and cultured ex vivo, have been shown to support influenza virus replication, as indicated by detection of infectious virus by titration and viral antigen via immunofluorescence staining.⁸⁰

The ferret lung consists of six lobes: left cranial and caudal, right cranial, middle, and caudal, and accessory (Figure 1B).Ferret lungs are generally uniformly pale pink and spongy, and variations in color and consistency are not uncommon in uninfected lungs. Collection of small pieces from each lobe (≤1 g in total) are pooled for titration or other downstream applications. Lung specimens may also be sampled and analyzed separately to investigate localization of virus. The lungs can then be fixed for histochemical evaluation by perfusing formalin into the lungs so the retention of expanded alveoli is achieved. A 1-mL syringe may be used to deliver formalin to the lungs via the trachea or large bronchi. Considering the importance of studying lung tissue and function during influenza virus infection, there are a large range of downstream applications and analyses frequently performed on this tissue.

Like the trachea, uniformity in collection of lung samples among groups is essential, as viral loads may differ between lobes of the lung, as well as spatially within each lobe.¹⁹ For example, collection of samples at the perihilar regions of the lung may yield more reproducible and detectable viral titers than samples collected toward the periphery of each lobe. Infectious virus titers and viral antigen have been shown to vary between the bronchus, bronchioles, and alveoli, depending on the virus inoculum and the day postinfection sampling occurred.⁸¹ Similarly, induction of proinflammatory cytokines and chemokines after infection may vary between different compartments of the ferret lung,⁸² necessitating uniformity in collection and reporting of the samples being examined. Live imaging of neutrophil levels in the ferret lung after influenza virus infection further supports regional differences in cellular infiltration, both globally and locally within this tissue at different time points after inoculation.⁶² The use of next-generation sequencing has improved our understanding of the variance in subsets of viral populations throughout the ferret respiratory tract, highlighting the contribution of virus replication at different anatomic sites toward virus pathogenicity and transmissibility in this model.^56,83

Gross lesions are frequently observed in lung tissue during acute influenza virus infection and warrant careful evaluation. However, an in-depth review of the lesions seen with influenza viruses in the ferret model is outside the scope of this review. Generally, gross pathology can vary from mild suspected pulmonary consolidation (≤10% of the lung) with viruses of low virulence to multifocal or diffuse consolidation and/or discoloration often associated with highly pathogenic viruses. The spectrum of histopathologic lesions of influenza viruses in the ferret is highly dependent on the virus strain. In general, both low- and high-virulence influenza A viruses infect the airway epithelium from the nasal passages to the bronchioles. Such lesions include necrotizing rhinitis, with or without tracheitis, bronchitis, and bronchiolitis. Highly virulent influenza A viruses typically exhibit injury of airway epithelium that, dependent on the virus strain, can extend into the alveolar septae/interstitium and/or extrapulmonary tissues. For example, neutrophilic and mononuclear inflammation can be observed surrounding bronchioles and spreading into the alveolar interstitium and spaces, accompanied by intra-alveolar edema and hemorrhage.^{46,71,72,79,84}

The draining lymph nodes (hilar and mediastinal lymph nodes) may be difficult to locate in uninfected ferrets but can be informative when assessing inflammatory responses during the acute phase of infection. Titration and immunohistochemistry have identified the presence of influenza virus in the ferret hilar lymph nodes,⁸⁵ and leukocyte populations have been analyzed in mediastinal lymph nodes.⁴⁹

Influenza viruses are a principal respiratory pathogen in humans; ocular tissue represents both a potential site of influenza virus replication and a potential anatomic conduit to establish a productive respiratory infection in mammals.⁸⁶ Ferrets have been identified as a suitable experimental model for research pertaining to the ocular system,^87–89 and infectious virus has been recovered from eye swabs and washes in ferrets.³⁸ Conjunctival tissue from the ferret may be obtained by using a scalpel to remove the tissue surrounding the opening of each eye (approximately 1-cm radius from the eyelid). The globe (eyeball) may be removed by proptosing it from the orbit and using scissors to transect the optic nerve and surrounding retrobulbar soft tissues (Figure 2). Conjunctival and eye tissue may be differentiated as right or left or pooled for collective conjunctival and eye samples.

Eyes and conjunctival tissue are infrequently sampled during necropsy of influenza virus–infected ferrets, but influenza viral antigen has been reported in the eyes and conjunctival tissue after ocular inoculation, and respiratory or ocular exposures to influenza virus.^38,90 Ferrets do not routinely develop conjunctivitis after either intranasal or ocular inoculation with influenza virus.⁶³ Proinflammatory cytokines and chemokines have been detected during the acute phase of infection in these samples.⁵³ To facilitate complete fixation of the relatively large globe, injection of formalin into the vitreous chamber through the sclera at the limbus may be performed to help preserve the delicate ocular anatomy and inactivate any virus that may be present.

Although influenza viruses are not typically neurotropic, influenza viruses exhibiting high virulence in ferrets may spread to the central nervous system via the olfactory and cranial nerve pathways, as well as via the inner ear and vestibulocochlear nerve.^51,64,91 Depending on the scope of the study, various methods for exposure of the brain may be required. Traditionally, removal of the entire calvarium using rongeurs can allow the whole brain to be obtained intact for subsequent sampling of specific areas of interest. Alternatively, removal of half of the skull using rongeurs can allow for lateral extraction of the brain, starting at the orbit to generate the opening (Figure 2). Brain tissue should be handled and sampled in a caudal to rostral direction as higher titers of virus are found in the more rostral regions of the brain, but the caudal and rostral cerebrum may be pooled if this is not a concern.⁹² The olfactory bulbs, located rostral to the cerebrum, can be removed from the calvarium while still attached to the cerebrum or separately. Infectious virus may be detected in olfactory bulb tissue in the absence of virus detection in the cerebrum; this is often attributed to the close proximity of olfactory bulbs to the nasal passages, where the highest viral load is typically detected in an infected ferret. Therefore, olfactory bulbs should be manipulated last as this region of the brain typically holds the greatest amount of virus from this organ.

Viral titers and viral antigen have been occasionally reported in the ferret cerebellum, brainstem, spinal cord, and cerebrospinal fluid after infection with highly virulent viruses, concurrent with histologic evidence of inflammation within these sites.^57,66,70 Detection of viral antigen has also been reported, albeit sporadically, in meningeal cells surrounding the cerebrum, cerebellum, and spinal cord, ependymal cells lining the ventricles, and astrocytes.^51,70,91 Inflammation seen by histopathology is not uncommon after ferret inoculation with highly virulent viruses.⁴⁵ Like the cerebrum, cytokine and chemokines can be measured in this tissue.^48,52 Remaining tissue is formalin fixed for histopathologic analysis and immunohistochemical staining.^57,64

Nasal turbinates can be accessed by using a scalpel to excise the nose and an approximately 1-cm segment of the muzzle, revealing a cross-section of the nasal cavity with the right and left chambers bisected by the nasal septum (Figure 2). Forceps are then used to dislodge and forcibly extract fragments of nasal turbinate tissue from both chambers. Because of the small total sample collected, nasal turbinates are collected in their entirety for subsequent processing, or the sample can be split to preserve tissue for concurrent immunohistochemistry assays.⁵⁷ Alternatively, removal of the overlying nasal bones can be performed using rongeurs to expose the entire nasal canal up to the ethmoid turbinates, allowing more precise anatomic localization for tissue sampling. Histopathologic and/or immunohistochemical examination of the entire nasal turbinate tissue can also be performed by fixation of the intact calvarium, after decalcification and serial sectioning through the entire nasal region.

Nasal turbinates typically contain high viral loads in ferrets infected with influenza virus, and, like the trachea, they provide important supporting information to interpret titers from nasal wash or swab specimens. Ex vivo infection of differentiated ferret nasal turbinate epithelial cells further supports the capacity for high titer virus replication in this tissue.^66,93 Expression of proinflammatory cytokines and chemokines may be measured in homogenized samples of this tissue.⁶¹ Furthermore, deep sequencing of viral quasispecies has been performed in this sample,⁵⁶ which can provide critical information when examining the emergence of viruses bearing mutations that facilitate mammalian adaptation.⁹⁴

The soft palate, located on the caudoventral aspect of the nasopharynx (Figure 2), bears permissive receptors for influenza virus and has been recently identified as an important tissue for host adaptation of influenza virus.^4,56 Visualization of the ventral aspect of the upper palate can be achieved by removal of the soft tissues between the mandibular rami, including the tongue, or via dislocation of the entire mandible at the temporomandibular joints. A scalpel or scissors can then be used to excise the soft tissue caudal to the hard palate. Soft palate tissue may support replication of high levels of virus; furthermore, inflammation of the soft palate after influenza virus infection can be observed by histopathology and immunohistochemistry.^56,95 The tonsils are not routinely sampled, but have been reported to exhibit changes in immunostaining patterns after influenza virus infection.^70,73 This tissue can be obtained at the time of soft palate excision. Leukocyte populations have been occasionally assessed after infection in medial retropharyngeal lymph nodes.⁴⁹