Limitations of infection detection methods used today

Limitations of infection detection methods used today

  • General comparison
  • Culturing
  • Rapid test kids
  • PCR
  • MALDI-TOF

General comparison

The methods widely applied for bacteria detection in veterinary practice are shown in Table 1. However, these methods are either unreliable (quick test), expensive (MALDI-TOF) or slow (culturing, PCR).

Table 2. Methods currently used for bacteria detection in veterinary practice.

Method Principle of the identification Advantage Drawback
Classical culturing in Petri dish[1] Morphology Standard, well-known, easy Time consuming (12-48 h), human factor in the measurement, sterile laboratory environment, price: 10-40 € per sample (depending on the bacteria and country of location).
Rapid test Colour change, due to pH change Relatively rapid, small, easy to handle, Not reliable (less than 60%), relatively expensive (10 €), single use only
Polymerase Chain Reaction (PCR) [2] DNS detection Accurate, small size, database is required. Relatively slow (12-24 h), expensive (50 €), dead bacteria are also detected (complicating factor)

MALDI-TOF/MS [3]

 

Structural analysis of proteins and their fragments a Rapid (1-2 hours), accurate Expensive equipment (300-500 thousand euros investment), high vacuum, high maintenance costs, difficult sampling preparation. Cost per sample is about 100 €

[1] In Hungary, these methods are standardized: MSZ–364/4–86, MSZ–- 3640/18–1979, MSZ–3640/12–197

[2] WO 2006/085948

[3] US 6177266

Culturing

Culturing of different bacteria in one petri dish

The most widely applied method is microbiological culturing. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both. It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease by letting the agent multiply in a predetermined medium. Microbiological cultures can be grown in petri dishes of differing sizes that have a thin layer of agar-based growth medium. Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the best temperature for the growing of the selected bacteria (for example, usually at 37 degrees Celsius for cultures from humans or animals, or lower for environmental cultures). Microbial cultures are foundational and basic diagnostic methods used extensively as a research tool in molecular biology. The identification of the bacteria is made upon visual identification, which makes the method unreliable. Determination of several bacteria at the same time meets also difficulties. But the largest drawback so far is the waiting time of minimum 12 hours, typically 48-72 hours. The test requires human intervention, and the cost is between 10 and 50 €/test, depending on the country (high labour cost).

 

Rapid test kid

Rapid test kid

Rapid test kids are usually based on specific reaction or reagent. Using this method only the dedicated bacteria can be detected, so the user must have a clear preconception about the possible disease. No rapid tests are available for all kinds of bacteria. Its major advantage is the short response time (few minutes), and the drawback is its low reliability. Nevertheless, its price is quite expensive, can reach 10 €/test.

 

 

 

 PCR

The principle of the PCR method

Polymerase Chain Reaction (PCR) is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies
of a particular DNA sequence. PCR permits diagnosis diseases. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity that is at least 10,000 fold higher than that of other methods PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses. PCR also permits identification of non-cultivatable or slow-growing microorganism
s. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes[4]. The main advantage of PCR is its high accuracy, small size.  Its drawback is that the response time is relatively slow (12-24 h), and also expensive (50 €). As a complicated factor dead bacteria can also be detected.

 

MALDI-TOF

MALDI-TOF spectrometer

Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) is a special type of mass spectrometry that can be used for rapid and reliable detection and identification of bacterial species[5].  MALDI
TOF are usually applied in diagnostic of humans and identification of different organism. In MALDI-TOF the high molecular weight DNA or protein species are identified. MALDI methodology is a three-step process. First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated gases, and can then be accelerated into whichever mass spectrometer is used to analyse them[6]. Its major advantage is its high speed (1-2 hours), high accuracy. On the other hand, the equipment is very expensive equipment (300-500 thousand euros), requires high vacuum, high maintenance costs, difficult sampling preparation. The cost of one measurement can be up to 100 €.

[4] Cai, H; Caswell J.L.; Prescott J.F. Veterinary Pathology 51 (2): 341–350.

[5] Silpak Biswas, Jean-Marc Rolain, Journal of Microbiological Methods Volume 92, Issue 1, January 2013, Pages 14–24.

[6]  Karas, Michael; Krger, Ralf (2003). Chemical Reviews 103 (2): 427440.

Antibiotic usage: regulations

Antibiotic usage: regulations

One of the important problems of humanity is to provide enough food for the increasing population. Production of meet with increasing efficiency and lower costs induced the spreading of intensive farming technologies. In particular, poultry (especially chicken) is grown at large scale under artificial conditions. These intensive technologies, however, resulted in increasing number of diseases due to infections (both bacterial and viral). In order to reduce the chance of bacterial infections antibiotics started to be added as additives to feedstuff. In The United States, Food and Drug Administration approved the use of antibiotics as animal additives without veterinary prescription in 1951. In the 1950s and 1960s, each European state approved its own national regulations about the use of antibiotics in animal feeds[1]. Later, several concerns arose about the preventive usage of antibiotics in the feedstuff. The risk concerning residues of antibiotics in edible tissues and products that can produce allergic or toxic reactions in consumers was proved[2]. Moreover, the use of antibiotics as feed additives in the long run can contribute to the development of resistant bacteria to drugs used to treat (both human and animal) infections. For this reason, the World Health Organization (1997)[3] and the Economic and Social Committee of the European Union (1998)[4] concluded that the use of antimicrobials in food for animals is a public health issue. Based on these findings, several European states prohibited the use of antibiotics in feedstuff in the late 90’s. Afterwards, the European Commission regulated the additives used in animal nutrition[5]. In the regulation 1831/2003, it is stated that antibiotics (other than coccidiostats and histomonostats) might be marketed and used as feed additives only until the end of 2005. Later, anticoccidial substances, such as antibiotics ionophores, were also prohibited as feed additives.

After 2013, in the Member States of the European Union, medical substances in animal feeds are limited to therapeutic use by veterinary prescription[6].

In the day to day farming practice, a veterinarian must examine sick or deceased animals and prove the bacterial infection before applying any antibiotics. This proof, however, requires bacteriological culturing under laboratory conditions, which takes 24 to 48 hours (up to 72 hours). During this time, unfortunately, several animals can be infected, resulting in significant economic losses for farmers and/or application of antibiotics to more animals and/or at higher dose.

Intensive farming of broiler chicken

 

[1] Jones, F. T. S. C. Ricke. 2003. Observations on the history of the development of antimicrobials and their use in poultry feeds. Poult. Sci. 82:613–617.

[2] Donoghue, D. J. 2003. Antibiotic residues in poultry tissues and eggs: Human health concerns? Poult. Sci. 82:618–621.

[3] World Health Organization. 1997. The medical impact of the use of antimicrobials in food animals: Report of a WHO meeting, Berlin, Germany.

[4] Economic and Social Committee of the European Union. 1998. Opinion on resistance to antibiotics as a threat to public health. No. ESC-98-016-EN.

[5] Regulation 1831/2003 of the European Parliament and of the Council

[6] Natasha Gilbert: Antibiotic resistance marching across Europe, Nature News, 22 November 2011

Chicken market

Chicken market

Chicken production is progressing all over the world. According to the World Watch Institute, 74 percent of the world’s poultry meat, and 68 percent of eggs are produced in ways that are described as “intensive”.[1] The USA is the world’s biggest producer of broiler chicken meat, 17,752 Million metric tonnes per year[2].  Brazil is the world’s second chicken grower with 13,115 MT followed by China (13,000 MT). In the case of the three leading countries as well as in others (like India, Russia) antibiotics may be used as additive to feedstuff to promote growth and avoid early death of animals. However, thanks to the strong media campaigns and opinion of professional community their usage is decreasing, in particular in the US. In 2012, FDA asked livestock and poultry producers to phase out use of antibiotics for growth purposes[3]. The American Meat Institute (AMI) and its members support FDA’s decision. However, no obligatory ban has been made yet.

Figure. Broiler meat production in the EU (2015)20

In Europe, the broiler sector is expected to continue to grow in 2016 and 2017. Production is also supported by strong export demand[4]. The overall EU-28 production in 2015 encompasses various situations, but broiler meat production is expected to increase from 2014 in major EU producing countries and especially in Poland which is on the verge of becoming the largest EU-28 broiler producer (Figure). In Germany, production will continue to grow but welfare and environmental issues are hampering faster growth in the industry. Production will however remain flat in Spain because of the pressure from the retail industry on producers negatively impacting producer margins. The significant and continuous decrease of grain prices in the EU-28 since 2013 has increased operating margins, as retail prices decreased less.20 Beside market trends, the increase in the prices for 2013 can be explained also by the implementation of straight ban of the use of antibiotics from 2013. In order to reduce the operational costs, users must reduce their costs, including costs for veterinarian services, costs of antibiotics, loss due to deceased animals.

[1] State of the World 2006 World watch Institute, p. 26

[2] http://canadiansmallflockers.blogspot.hu/2015/04/chicken-exporters-for-world.html

[3] American Meat Institute report (2013): The Facts about Antibiotics in Livestock & Poultry Production.

[4] Xavier Audran, David G Salmon: EU-28 Poultry and Products Annual, Global Agriculture Information Network (2015)