Histopathologic Assessment of Gut for Poultry Production and Applied Research

Published on: 10/6/2017
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The ongoing transfer of science and technology is a defining feature of the efficiency and success of commercial poultry production. Meeting the nutritional, environmental, and health needs of every bird is key to realizing genetic potential and providing for well-being. To monitor and quickly identify developing problems, basic knowledge and skills in pathology are learned by almost everyone involved in live production. To add value to the standard postmortem procedure, we describe a method of applying histopathology (microscopic pathology) to digestive health surveys in broilers, turkeys, and commercial layers and to field and floor-pen research trials. 
In developing this method, principles were borrowed from toxicologic pathology as well as pharmaceutical and biological safety testing. In these fields, testing is performed in laboratory animals of defined genetics that are kept in controlled environments and fed uniform diets.  Within these systems, the disease is defined as any departure from normal caused by a known toxin, product or infectious agent.    
Pathology is a key part of the assessment process, performed at specific times or ages after exposure. The assessments are carried out using standard operating procedures that lead to thorough examinations of either all body systems, or perhaps narrowed to target organs.  The pathology assessment includes recording of any lesions observed at necropsy (animal autopsy, postmortem examination), and lesions that are detected by histopathology.   
Fundamental to the pathology examination are 1) presence or absence of a lesion; 2) the lesion severity; and 3) assessment of incidence and severity differences from expected normal for the test population. The latter takes into consideration age, genetics, and spontaneous pathology, and/or comparisons to internal controls in the study.  The process becomes progressively complicated as multiple lesions are found, each requiring assessment according to standard procedures for scoring severity with analysis to determine significance.  It is labor intensive and detailed work performed by a pathologist. Because the process is standardized, however, the results are reproducible (if the protocols are followed) even when the analysis is conducted by independent parties. It also allows development of data sets that can be monitored over time for changes in disease expression within a group or between groups.    
From a broad perspective, commercial poultry fit this model reasonably well: animal populations of uniform genetics, consuming a uniform diet, and raised under relatively uniform housing and management programs with good to excellent biosecurity.  From any production program, it is possible to select birds of specific ages, on specific management or nutritional programs, and make a uniform pathologic assessment according to standard procedures, and obtain reasonably reproducible results by persons trained in the evaluation methods.
In fact, this is already well established as “posting sessions” that serve to populate a database for tracking gut pathology over time. The Health Tracking System (HTS®) (Elanco, Greenfield, IN, USA) is a retrievable database for recording gross (macroscopic) pathology of the gut. We have endeavored to go to step deeper in the understanding of events in the digestive system by integrating histopathology into the gut evaluation process.  
The traditional application of histopathology involves follow-up to a necropsy involving a disease outbreak (1) primarily for confirmation of a specific disease (coccidiosis, necrotic enteritis, histomoniasis) occurring in one or several flocks and meriting immediate treatment.   Integration of histopathology for assessing gut health differs from this approach in that it usually involves clinically normal or representative birds from a program, and looks for deviations in normal structure that may underpin or contribute to a disease process.  This systematic assessment seeks to identify abnormalities in the gut, measure the severity of the observed factor, and mark the duration of each factor.  Examples include the presence of an organism (coccidia, bacteria), a lesion, or an atypical material in the gut lumen (insect exoskeleton, foreign material from a problem ingredient).  
To be practical, the application of histopathology to production must have sufficient detail to identify key events in gut pathology without succumbing to tedious detail more fitting to basic research. For this reason, the method uses semiquantitative scoring (graded categories) versus quantitative measurement (counting or measuring specific parameters). From a single biological specimen, key lesions are assessed, scores are assigned, and data is recorded in a spreadsheet. From this dataset, various indices (digestive, immune) are calculated: for individual lesions and for clusters of lesions. The indices can be graphed or further analyzed to yield a summary of what is happening when it happens, how bad it is (peak severity), and how it plays out over time (the sequential appearance and overlap of the individual lesions).   

General Guidelines. A typical survey would examine 5 birds each at 3 to 4 day intervals from about 12 to 35 days of age or older, depending on the focus of the survey. Five birds represent the number of tissues that will fit on a histologic slide. For example, examining three levels of gut (duodenum, jejunum, cecum) would require three slides for five birds.   
Depending on the situation, a survey could include all or any combination of proventriculus, ventriculus (gizzard), duodenum with pancreas, jejunum, cecum with ileum, and liver. Lesions in these tissues are included for the basic profile because they, 1) influence digestive form and function, 2) represent partitioning of nutrients from growth to inflammation and repair, and 3) lend themselves to rapid assessment with a severity score.  Multisystemic surveys might also include bursa of Fabricius and thymus, or bone. Surveys also include assessment of parasite loads.   
Examples of commonly collected tissue combinations reflect the problems observed in a program, such as jejunum only; duodenum, jejunum and cecum; proventriculus, duodenum, jejunum, and cecum; and variable inclusion of liver with any of the above sets of tissue. Gizzard and ileum are examined infrequently in production surveys. Bursa of Fabricius is commonly included in gut surveys; thymus, kidney, and trachea can be added.
Lesion scores are 1, normal; 2, mild; 3, moderate; and 4, marked to severe. This scale is arbitrary and the system also works well with a 0 to 5 scale (normal, minimal, mild, moderate, marked, severe, respectively). Each score for each lesion is defined in a standard operating procedure. Indices are calculated by summing total scores and removing normal scores, with the remaining index representing a lesion burden. For coccidia and intraepithelial lymphocytes, the scores represent a range of increasing numbers of organisms or cells.  
Almost all production surveys are read and reported to the client without knowledge of the feed additive or vaccination programs. In field trials, it is known that products are being compared, but the specimens are coded to blind the observer. In floor-pen research, pen number and bird number are known but treatments are coded. 
Surveys may reveal lesions of specific diseases in clinically normal birds, such as coccidiosis, necrotic enteritis, histomoniasis, viral enteritis (runting stunting syndrome). Equally important, numerical data is also accrued on the necropsy category of nonspecific enteritis characterized by short villi, crypt hyperplasia, increased intraepithelial leukocytes, heterophilic inflammation, and dysbacteriosis (4).  
Morphometric measurements of intestinal villi and crypts can be added to production surveys, using the same sections as used for the histopathology assessment, and are measured in the duodenum and jejunum. Five villi and five corresponding crypts are measured in each section of gut. The mean villus: crypt ratio is calculated and the mean mucosal thickness is calculated. 

Results. Several trends have emerged in surveys conducted in broilers. 
• Lesions of viral enteritis occur in many young broilers. The lesions are of mild (likely subclinical) to moderate severity (possibly clinical), and may be present as late as 24 to 28 days of age.
• Eimeria maxima is the dominant coccidia species with variable replication age range, but uniformly peaking in incidence and severity at 25 to 26 days of age. Minor strains of coccidia are detectable and may be prevalent in some complexes.
• Coccidia vaccine oocyst replication can be monitored in surveys modified for this purpose.
• The ileum is relatively quiet from a pathology perspective in contrast to duodenum and jejunum, but may show dysbacteriosis and minor strains of coccidia.
• Intraepithelial leukocytes are a variable presence in the small intestine and may be an indicator of intercellular tight junction integrity.
• Although the cecum is a bacterial fermentation organ, actual adherence of bacteria to the surface of cecal enterocytes is variable. Of interest is the occurrence of adherence with micro-erosion of the mucosal ridges and lymphoid apoptosis and heterophil infiltration of the lamina propria supporting the mucosal ridges. These changes may have a role in systemic bacterial dissemination from the gut.
When combined with surveys of the immune system, the gut issues can be seen from the perspective of lymphoid organ stress associated with viral enteritis, the effect on the bursa of Fabricius of acute and recovery stages of infectious bursal disease, and thymus atrophy from infection by chicken infectious anemia virus following decline of maternal immunity (2, 3).
In commercial layer pullets, interesting findings include lesions suggestive of viral enteritis, enteritis related to feed ingredient quality, and coccidia cycling patterns. In layers, coccidiosis, focal duodenal necrosis, midgut dysbacteriosis, and cecal spirochetosis can be observed.  
In turkeys, lesions consistent with viral enteritis, coccidia, dysbacteriosis, other protozoa (cryptosporidium), and the development of cellular inflammatory components in the lamina propria can be seen.  
Field trials for new products have been evaluated using this method. In floor pen trials, the standard production lesion profile is used, frequently with the addition of villus and crypt measurements. Other quantitative measures can include the enumeration of specific cell populations. In these cases, the study is read with knowledge that products are being compared, but the specimens are coded to minimize bias in the process. For floor-pen trials, the tissues from one bird are placed on a slide, providing individual bird data for analysis.   
Technical Guidelines. The histopathology lesion scoring process is labor and time intensive and requires specimens of the highest quality. Five birds per group is appropriate and allows the efficient and economical placement of five similar tissues on one slide. If sampled as sequential ages, five per group provides enough data to define disease progression, while keeping the size of the study manageable in terms of expense and turn-around time. The following technical guidelines (5) help to ensure a successful survey.
• Collect specimens within 3 to 5 minutes of euthanasia;
• Collect tissues from uniform tissue locations;
• Use 10% buffered formalin for tissue fixation (diluted hatchery formaldehyde is unacceptable).
• The final volume of formalin is preferably 10 parts formalin:1 part tissue (a ratio below 5:1 is unacceptable);
• Handle the tissue gently;
• For gut, keep sections closed and 2 cm maximum length;
• Avoid freezing tissue for histopathology (destroys the tissue detail needed for analysis).
• 90% of formalin can be removed prior to shipping – 10% remaining to keep tissue moist;
• Specimen jars that leak before shipping will leak during shipping; use secure containers.
Prefilled surgical buffered formalin containers are recommended for specimen collection in the field. Containers with screw-cap lids that can be securely tightened to avoid leakage are best. For routine use, 180 ml containers (containing 90 ml buffered formalin) are a good choice.
References and Links 
1. Abdul-Aziz, T., Fletcher OJ, and HJ Barnes. Avian Histopathology, 4th Edition. American Association of Avian Pathologists, Jacksonville, FL. 2016.
2. Hoerr, FJ. Invited review: Clinical expression of immunosuppression in poultry. Avian Diseases, 54:215, 2010.
3. Hoerr, FJ. Multifactorial diseases of broiler chickens. American College of Veterinary Pathologists, Proceedings CD. 2011.
4. Hoerr, FJ. Assessing Gut Health with Histopathology. Multi-State Poultry Feeding and Nutrition Conference, Proceedings CD, 2013. www.vetdx.com.
5. Hoerr, FJ. Digestive Survey Tissue Collection Guide; 2015. www.vetdx.com.
Presented at the 2016 Arkansas Nutrition Conference and published in the proceedings.
Dr. Hoerr was a diagnostic pathologist with the Alabama state diagnostic laboratories for 32 years, and served as director of laboratories from 1987 until his retirement in 2012. During this time he held a joint appointment on the faculty of the Auburn University College of Veterinary Medicine where he continues to participate in poultry disease research and teach as Professor Emeritus. Frederic J. Hoerr, DVM, MS, PhD, who is board certified in veterinary pathology and poultry medicine provides Histopathologic examination at the Veterinary Diagnostic Pathology, LLC- Visit http://www.vetdx.co
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