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Gut Health and Diagnostic Challenges Addressed at AVMA Convention

08 October 2013

One session at the 150th annual convention of the American Veterinary Medical Association (AVMA) in Chicago in July 2013 was devoted to poultry gut health problems and associated diagnostic issues.

Morphometric and Functional Analysis for Assessing Poult Intestinal Health

With feed accounting for majority of production costs, maximising intestinal function becomes increasingly important, said Takumu Niino of North Carolina State University's College of Veterinary Medicine. Currently, there is no “gold standard” for assessing the physiological function and integrity of the intestine. In many species, including poultry, absorption of nutrients, water, and electrolytes takes place primarily in the small intestine; therefore, any insult to this region can cause malabsorption and subsequent increased FCR.

The objective of the study, Dr Niino reported, was to examine the relationship between morphometric analyses and results from the Ussing Chambers, an apparatus that measures transcellular ion movement and paracellular carbohydrate movement, in order to assess potentially impaired nutrient absorption in the jejuna of commercial hens that were average in weight ('normal') and those that were substantially smaller ('stunted').

Absorption of nutrients is ultimately mediated by the energy dependent movement of sodium ions across the intestinal epithelium. This process creates an electrochemical gradient, followed by the movement of nutrients and water. Impairment of this process may lead to improper nutrient absorption and an increased feed conversion ratio.

The Ussing Chamber has been used to determine the integrity and functional capacity of gut epithelium by measuring Transepithelial Resistance (TER) and Paracellular Flux (PF) of mannitol.

Ultimately, explained Dr Niino, TER reflects the integrity of the gap junctions, which indicates how 'leaky' or permeable the intestine is. PF measures passive diffusion of mannitol across the epithelial membrane.

A higher TER suggests that the epithelial membrane is more tightly bound, hence providing more resistance to ion movement. A high mannitol flux indicates the gut epithelium is more permeable to larger molecules.

In-vitro Antimicrobial Susceptibility of Clostridium perfringens of Broiler Origin

Clostridial enteritis is a common disease affecting the intestinal health of many US commercial broiler flocks. Jaime Ruiz of Phibro Animal Health - North American Region explained that several antibiotic feed additives have been used to control this costly disease condition.

Field isolates of Clostridium perfringens were collected between 2011 and 2012 at several broiler production complexes located in the Southern United States. These isolates were tested for their ability to produce visible growth in the presence of antibiotics on a series of agar plates containing dilutions of the antimicrobial agent (agar dilution).

He presented updated information regarding Minimum Inhibitory Concentrations (MICs) against virginiamycin and other commonly used antibiotic feed additives in his paper.

Evaluation of the Efficacy of Various Feed Additives on Broiler Chicken Performance Parameters using a Controlled Clostridium perfringens Challenge Model

Clinical Necrotic Enteritis (NE) is a clostridial toxin-related disease that results in extensive, full-thickness necrosis of large areas of the small intestine, according to Blair E. Telg of Elanco Animal Health. A separate intestinal disease, clinical coccidiosis, arises when lesions associated with one or more of the coccidial parasites are present within the intestinal tract of an infected chicken. Lesions associated with clinical coccidiosis can predispose a chicken to develop NE.

Challenge models have been developed to experimentally replicate what is observed in field outbreaks of clinical NE.

In this trial, five treatment groups consisting of 360 Cobb 500 males per treatment group were used. All treatment groups, except the unchallenged control group, were challenged with Clostridium perfringens at 17 days of age.

The objective of the study was to compare the effects of various feed additives on broiler performance when delivered ad libitum in feed to broilers challenged with Clostridium perfringens in a controlled NE challenge model.

The NE challenge model that was used in this study resulted in more than 30 per cent NE-related mortality in the challenged, no-additive control group.

Dr Telg reported that all groups that received an in-feed additive had significantly lower mortality and significantly better weight gains and lower feed conversions at the end of the trial (day 24) than the challenged, no-additive control group following the Clostridium perfringens challenge on day 17.

Comparison of the Intestinal Microbiota Effects of Bacitracin Methylene Disalicylate (BMD) and Virginiamycin (Stafac) in Commercial Broilers

FDA-approved antibiotics are administered via feed to commercial broilers to promote intestinal health and function, resulting in improved weight gain and feed efficiency. However, the exact mechanism for how this improved performance is achieved remains unclear, reported Anthony Neumann from the University of Wisconsin–Madison.

The purpose of the study he reported, conducted with Colorado Quality Research, Inc. was to determine the effects of two commonly used broiler in-feed antibiotics, bacitracin and virginiamycin, on the jejunal and caecal bacterial community composition of mature broilers using state of the art, high-throughput DNA sequencing.

Gastrointestinal tracts were dissected from Cobb 500 broilers at 45 days post-hatch, and samples taken for DNA extraction and subsequent 16S rRNA gene targeted amplicon pyrosequencing.

Additionally, Clostridium perfringens-specific quantitative PCR was used to determine the effects of the respective feed additives on the jejunal and caecal populations of this important poultry pathogen. A total of three subsequent simulated grow-outs were sampled and analysed.

Bioinformatics and statistical analysis of the resulting sequencing data and qPCR observations and treatment correlations were discussed.

Discussion of the European Food Safety Authority's Process for Evaluation of Zootechnical Feed Additives and Possible Application in the US Poultry Industry

As interest has increased in alternatives to traditional antibiotic feed additives, the US poultry industry has seen an impressive rise in the number of products that are being marketed based on claims that are beyond the scope of the products’ labels, according to Patrick M. Pilkington of Vetagro, Inc. These claims are based on research of varying quality leaving the end-user, often a veterinarian and/or a nutritionist, with the responsibility for proper evaluation. The task of proper evaluation is often overwhelming and the need for a third-party evaluation system is evident.

While the US industry would not welcome a comprehensive ban on the use of antibiotics in animal feed as adopted by the European Union, Dr Pilkington said that the rules that subsequently were set out for authorisation, supervision and labelling of feed additives by the European Commission are worth exploring. Such rules may provide the third-party evaluation of feed additive products that is needed by those responsible for the nutrition and health program of the US poultry flock.

Dr Pilkington's presentation described the current overwhelming task of product evaluation faced by today’s poultry veterinarians and nutritionists, introduced the European Food Safety Authority’s (EFSA) role in such evaluation in the EU, and outline the EU approval process by describing the history of one of the first products to gain approval as a zootechnical additive, namely Aviplus P.

Aviplus P is a microencapsulated blend of purified botanicals and organic acids manufactured by Vetagro, S.p.A and currently marketed in the US by Vetagro, Inc.

Optimisation of Carboxy-X-Rhodamine Dye (ROX) Concentration for Real Time RT-PCR and Plus/Minus Assays using TaqMan® Probes Chemistry; Implications on Molecular Diagnostics of Avian Pathogens

Carboxy-X-rhodamine (ROX) is a passive internal reference dye used to normalize real-time RT-PCR and PCR assays, explained Hayet Abbassi of the University of Minnesota, St. Paul, adding that it is used to compensate for non-PCR related variations in fluorescence and changes between wells caused by slight volume differences or due to the design of the instrument.

ROX reference dye is believed necessary for use in combination with TaqMan® Probe Master on Applied Biosystems (ABI) real-time PCR instruments in order to use readily its default parameter analysis. This dye is included in most real time RT-PCR commercial kits, either in the core reagent kit or separately for additional flexibility with various instruments.

Most of the molecular diagnostic assays performed in the Minnesota Veterinary Diagnostic Laboratory (MDVL) use the TaqMan® chemistry and the ABI instruments. The widely held position by major diagnostic laboratories and the industry is to respect the recommended concentration of the reference dye because changes can lead to unintended consequences without any bearing on the sensitivity of the reaction.

However, Dr Abbassi's experiments using ABI 7500 real-time PCR system yielded results that challenge this commonly held knowledge.

The St. Paul team conducted several experiments for the molecular diagnostic of aMPV by real-time RT-PCR and Plus/Minus Assays using various concentrations of ROX reference dye and comparing different commercial kits from various companies.

These experiments involved virus isolates, clinical, research and internal samples. The results showed that the recommended ROX concentration used routinely in the MDVL were not adequate for the molecular assays with the ABI 7500 system.

The findings suggest that ROX concentration can affect the sensitivity of molecular diagnostic assays and the level of discrimination between positive and negative samples. Although this passive dye does not participate in the PCR reaction, its concentration seems to have an effect on the outcome of the molecular diagnostic results.

As a result of the study, Dr Abbassi recommended the optimisation of the concentration of this passive dye during the design of every molecular diagnostic assay and/or the use of different real-time PCR instruments.

Further Reading

Find out more information on the diseases mentioned here by clicking here.
To read the previous article from the AVMA convention, click here.

October 2013



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