Assessing Gut Health with Histopathology

Histology is the normal microscopic anatomy of organs and tissues. Histopathology is microscopic pathology, involving altered structure, composition and function of organs and tissues.  Pathologists identify organ and tissue abnormalities at the macroscopic, microscopic, and molecular levels in order to diagnose disease and define the mechanism by which disease may be occurring (pathogenesis).

Many diseases of poultry have been identified, characterized, and largely controlled in the past 100 years, which contributes substantially to the success of poultry production today. These diseases are diagnosed by observing patterns of clinical signs in live poultry, and identification of lesions (abnormal tissue) at necropsy. Some lesions are more specific than others, and there may be uncertainty about whether an organ is normal or abnormal. Histopathologic examination of the tissue helps to resolve these situations, and is often performed with other ancillary testing, such as bacterial or fungal cultures, virus detection, serology, and toxicology.  The diagnosis of one or more diseases involves the collective assessment of this information. This is the standard diagnostic work-up in poultry production with the intention of finding diseases that can be treated or, preferably, controlled and prevented.

A health program that depends solely on identifying causes of mortality however is almost certain to remain behind the leading edge of problems. Poultry health surveys, which monitor not only mortality, but also examine clinically normal poultry for minor lesions and parasite loads, can help to define subclinical disease that erodes health and well-being, and profitability. Histopathology adds informational value to necropsy sessions.  In a research setting, a defined number of animals are selected for examination at specific times after treatment. This approach has been widely used in toxicological pathology and underpins pharmaceutical research and development. Because of the uniformity of the poultry production system, this same approach can be applied to vertically integrated programs.  The focus of this article is gut, but this method is applicable to the immune system, respiratory tract, or skeletal system – wherever a comparison is needed to understand a problem. It follows a research-type protocol that collects specific tissues from a defined number of animals (typically five), at defined ages and treatments, and selects tissue from consistent locations in the gut. Tissues must be collected in neutral-buffered formalin within 5 minutes of euthanasia to prevent autolysis; the tips of villi are especially sensitive. Extended collection times result in decomposition (autolysis) that diminishes the accuracy of the scoring. Sections of gut should be 1 inch or less in length for optimal fixation and left intact (not opened) to aid the consistent scoring of lesions.

The tissues are then sent to a histology laboratory and processed into slides. This uses paraffin-embedded tissue processing, with tissues cut on a microtome to 4- to 6 microns in thickness, and stained to aid in identifying tissue and cellular morphology. Although the rudiments of histopathology are taught in veterinary school, pathology is a specialty that requires three or more years of training to achieve basic competency. Poultry diseases are part of the training of veterinary pathologists but interpreting the results for decisions in vertically integrated poultry production requires additional experience. This encompasses lesions of specific diseases, lesions of less specificity that may be eroding performance, lesion severity, and the age of onset and resolution. This information can guide the development of strategies for intervention and prevention. For example, necrotic enteritis may the presenting problem, but a histopathology survey can assess the involvement of coccidiosis, viral enteritis, or less specific lesions that may reflect ingredient quality, natural toxins, or feed mixing errors. Each represents an intervention point that may help to reduce the incidence of necrotic enteritis. 

For gut, the primary sites for examination include proventriculus, duodenum and pancreas, middle small intestine (jejunum) at the level of the yolk stalk diverticulum, and mid-cecum. Depending on the problem, gizzard, lower small intestine (ileum), liver, and the immune organs (bursa of Fabricius and thymus), may also be examined. In broilers and turkeys, a typical survey might examine five clinically normal birds per flock at weekly intervals beginning at 7 days and extending to 28 days or older. Detailed histologic examination of the tissue is a time-intensive activity. The difference between basic research and production applications involves some compromise. The goal is to have enough detailed information to explain performance variability while keeping the process timely enough for practical usage.

The following parameters are typically examined for each tissue. For a routine survey, proventriculus mucosa and glands are examined and scored separately for inflammation and other lesions. For gizzard, the mucosa is examined for inflammation and other lesions, and the koilin lining for formation defects (erosion).  For duodenum, jejunum, and ileum, routine parameters are: coccidia load and species, villus length, crypt depth, cystic crypts, lamina propria cellularity involving gut associated lymphoid tissue (GALT) and other inflammatory cells, presence of bacteria on tips and sides of the villi, and “other” which includes intraepithelial leukocytes, excessive mucus, and other parasites. For cecum, the lesions scored are coccidia, other protozoa, GALT and lamina propria inflammation, bacterial adherence, cystic glands, and other. For each section of gut, each lesion is scored as 1, normal; 2, mild severity; 3, moderate; and 4, marked to severe. Definitions of each lesion and score provide consistency that allows for data banking and comparisons of surveys over time. Various lesion means and indices can be calculated for comparison. By using color coded conditional formatting in Excel, it is sometimes possible to observe diseases develop and resolve as colored clusters of individual tissue lesion scores across a spreadsheet.

The mucosae of the proventriculus and gizzard are commonly inflamed, a nonspecific lesion that is likely caused by several dietary stressors. The continuity of the gizzard koilin lining is usually a reflection of this. In broilers, transmissible viral proventriculitis is relatively common and produces glandular inflammation that may be missed at necropsy.

For the intestine, coccidia cycling can be detected by histopathology at levels below macroscopic observation. Infections by mixed species are common, and infections by atypical species can be detected and are of interest. Villus length is a general indicator of nutrient absorptive capacity of the gut. The crypt depth (hyperplasia) is an indicator of the degree of enterocyte replacement activity. It generally signals increased rates of cellular turnover with the potential for impaired enterocyte differentiation and digestive function. The crypts are secretory and crypt hyperplasia implies increased release of water into the gut lumen. Crypt hyperplasia is the most consistent lesion of enteric viral infections, and some crypts may become cystic. The intestinal lumen holds bacteria of mixed morphology, but the appearance of bacterial colonization and adherence to the tips and especially the sides of the villi is an indicator of dysbacteriosis and nonspecific bacterial enteritis.  The degree of lymphocytic and inflammatory cell infiltration of the lamina propria is an indicator of response to antigenic and inflammatory stimulation, respectively.  Intraepithelial leukocytes are an additional marker of these events, which may be accompanied by increased amounts of mucus along the sides and at the tip of the villus. The lesion scores can be variously combined, analyzed and indexed to provide semiquantitative indicators of impaired nutrient utilization and nutrient partitioning away from growth and into inflammation and repair. This is effectively moving research methods to the field for real-time assessment of gut health.

Additional specific measurements can be made of villus length, crypt depth, and the calculated villus-crypt ratio. Depending on the microscope, camera, and software, this can be done real-time on the actual field of view, or the measurements are made on digital photographic images of the gut section.

Histopathology surveys of gut are designed for general or specific purposes, and a survey consistently applied can provided comparative data over time. The applications include the comparison of management systems, coccidia control programs, feed additives, and alternative ingredients, as well as investigation of uniformity issues and farm performance.  In most surveys, there are lesion patterns that provide insight into issues of concern. It is relatively common to detect an unexpected disease or lesion. This provides objective data to correlate with performance, and to formulate prevention or control strategies. The process can be defined in standard operating procedures (SOPs), and performed under good laboratory practices (GLP).

Summary

Histopathology is a valuable diagnostic procedure that is applicable to routine diagnostics and to health surveys in poultry production. It can provide objective information about specific diseases and lesions, the severity of each lesion or lesion cluster, as well as the age of onset and resolution. This information is applicable to management decisions for disease prevention and control and provides semiquantitative data to assess nutrient loss and partitioning for inflammation and repair. Histopathology can be used to assess the influence of dietary ingredients as well as feed additives for health and performance.