Diagnostic testing has come a long way in the past 15 years. More sophisticated tests and procedures have enabled diagnostic laboratories to provide veterinarians and their clients with more accurate results in a shorter amount of time. But there are keys to getting the most out of bacteriologic testing – and it primarily falls back to sampling, which is generally under the veterinarian’s control.

Lorraine Hoffman, PhD, section leader in bacteriology at the Iowa State University Veterinary Diagnostic Laboratory, indicates that the most common enteric pathogens in cattle less than 2 months of age are E. coli, Salmonella species, Clostridium species, Cryptosporidia, rotavirus and bovine coronavirus. Causes of enteric diseases in animals greater than 2 months of age include Salmonella species, Clostridium perfringens, Coccidia and bovine viral diarrhea virus (BVDV).

Common pathogens associated with respiratory diseases are Mannheimia hemolytica, Pasteurella multocida, Histophillus somni (Haemophilus somnus), IBR, BVDV, PI3, BRSV, Mycoplasma bovis and Mycoplasma dispar. Although enteric and respiratory disease are the most common diagnostic requests at the ISU laboratory, pinkeye, diseases of the nervous system, systemic problems and abortions are also frequent submissions.

 

(Photo credit: Ray Grover)

Moist and cool are a must
The most important thing to keep in mind when collecting tissues and other samples from animals for bacterial diseases, especially for those animals that have died or have been euthanized, is that the samples must be kept moist and cool, and transported to the lab as soon as possible. “Bacteria generally thrive in a moist environment, so keeping the samples from drying out, maintaining them in cool temperatures, and speed are critical to ensure the greatest potential for recovery of the agent in question,” says Hoffman. This is necessary because postmortem contaminants or “normal flora” – if such exist – may overgrow the pathogen(s)     if allowed to remain at room temperature or above for extended periods.

“Samples that are dried out, such as swabs or small tissue pieces, are inconsistent in yield of pathogens.”

“The success rates from bacterial culture are improved by shipment of the samples in an environment that will both maintain the viability of the pathogenic organisms and prevent proliferation of contamination or decomposition of organisms,” adds Leann Siedlik, AS, Diagnostic Manager, MVP Laboratories, Inc., Omaha, Neb.

Time is of the essence, as well. Animals that have been dead for more than four hours will yield less satisfying results from bacterial sampling because they deteriorate very quickly post-mortem, particularly in warm temperatures. Organisms from the peritoneal cavity will invade other tissues rapidly. “The sooner tissues can be separated, the greater likelihood of successful culture,” says Siedlik. “Tissues from animals dead for more than four hours can be cultured, but the submitting veterinarian should be aware that the results may not be optimal.”

Other timing factors can influence your samples, as well. Submitting samples from animals that have already been treated with antibiotics will produce unsatisfying – and misleading – results. Treatment often reduces bacterial pathogen to low ‘antigen’ load and makes bacteria more difficult to recover. If you collect before treatment, you are giving the microbiologist the optimal chance to recover important agents.

Sampling “chronic” cases is often a lesson in futility, says Hoffman, because the agents left over causing the chronicity may not be the primary player. Secondary organisms that may not be associated with the acute infection may be recovered, such as Arcanobacterium pyogenes, but the pathogen responsible for the disease may no longer be present at the site.

 

(Photo credit: Iowa State University) Lorraine Hoffman, PhD, says sampling chronic cases is often a lesson in futility.

Avoid contamination
Contamination is one of the quickest and easiest ways to ruin a sample for bacteriological testing. Though food animal veterinarians often are not in optimal hygienic conditions for sampling, there are measures they can take to ensure the cleanest sample possible.

Good aseptic techniques should be practiced when collecting samples, both to protect the integrity of the sample and prevent exposure to the individual collecting the samples. Instruments used to collect samples should be as clean as the circumstances will allow. “Most collection situations can be adequately accommodated if common sense is used to guide the process,” says Siedlik.

Hoffman notes that aseptic techniques are more important than ever for maximum validity and accuracy because of the more sophisticated and sensitive testing methods available today. “For example, you should never use the same glove when collecting several fecal samples for Johne’s testing,” she says. “You should not lay a nasal swab on a dirty surface while you look for the mailing tube, you should always use clean towels when cleaning udders, etc.”

Milk samples are commonly collected for mastitis testing, but left to untrained individuals, sampling can be a waste of time and money. Untrained personnel may not understand the value in cleaning the udder thoroughly, releasing some milk before the sample is collected and keeping samples cool from time of collection to mailing. “It is my opinion that the veterinarian should be involved in the process so he or she is aware of the conditions and timing of sample collection,” says Hoffman. “At least they should train the collector in proper technique. Contamination can certainly cloud the interpretation of results.”

 

(Photo credit: Ray Grover) Swabs properly shipped, labeled and iced.

Siedlik adds that contamination of milk samples generally occurs when the milk is compromised by contact with the person taking the sample, the area surrounding the teat or with the use of a non-sterile collection container. Untrained individuals may have difficulty avoiding exposure of the milk sample to these and other possible sources of contamination. Milk samples should be collected in sterile snap-cap or screw-cap tubes, cooled and mailed with ice packs. Samples can also be frozen.

Use the right tools, containers
Avoiding contamination goes hand-in-hand with using the right sampling tools and containers. Hoffman says frustration abounds when veterinarians are called to post animals, collect nasal swabs or feces or draw serum from animals but do not have the correct tools or containers. It’s best to be prepared at all times with the recommended supplies. That can be accomplished by having one of the office personnel call the diagnostic labs(s) with which they work to obtain the list of commonly used tubes, Whirlpaks, etc. She notes that often this information is contained on the lab’s Web site if a veterinarian is unable to call the lab during office hours.

 

(Photo credit: Ray Grover) A biopsy sample that has been properly sealed and shipped in a biohazard bag.

The use of swabs for collection is a good example. Organisms die off more readily when on swabs, and contamination of swabs is an issue. Failure to handle the swab according to directions can result in inadequate or misleading culture results. Hoffman and Siedlik say that moist swabs in transport medium, such as Culterettes, are available, but care must be taken in their use to avoid contamination.

If you must use swabs for nasal, eye or vaginal sampling, Hoffman recommends the use of transport medium, keeping samples cool and transporting them quickly. Tissues or lung lavages are always a better alternative, but if the animal is not dead or if large numbers of animals need to be sampled, then the obvious is to collect feces or utilize a swabbing method.

 

(Photo credit: Ray Grover) Formalin leaking into the box is a potential biohazard

The primary sampling container, whether it’s a Whirlpak® bag, tube, etc., must be leak-proof. Samples in transit in the mail to diagnostic labs are subjected to harsh handling. Poorly packaged samples will leak through shipping containers. “This frequently causes unnecessary delay in delivery to the diagnostic lab,” says Siedlik. “It becomes necessary for the shipping agent to pull the shipping container from its regular delivery flow, bag it, and at times requires personnel from the receiving laboratory to retrieve the package at a local delivery station. Simple safeguards will prevent this from happening.”

Get to know your lab
One of the most important services you can offer clients is diagnostic testing, therefore having a good relationship with the lab(s) you use can help you make better sampling decisions and get more accurate information about the pathogens you’re dealing with. The sample-handling process at all laboratories is unique to that individual laboratory, and the work flow and personnel staffing can impact the processing of the sample. Knowledge of a laboratory’s capabilities, packaging preferences, sample preferences, days/hours of operation, etc., will enhance the satisfaction of the veterinarians with the results he/she ultimately receives.

“As an example, our laboratory specialized in the isolation and identification of mycoplasmas,” explains Siedlik. “We receive many samples based on the veterinarian’s desire for this service. We do not, however, offer histopathology. Consequently, when we receive fixed samples, we must contact the veterinarian for instructions on forwarding the samples to another lab.”

Knowing your diagnostic lab also involves being familiar with the types of tests that can be performed. Hoffman says within the past several years, two highly specific and sensitive testing procedures have revolutionized the veterinary diagnostic world. These techniques, which do not rely upon isolation of the agent (which in some instances is challenging, especially when dealing with viruses and Mycoplasma species), are immunohistochemistry (IHC), which is the microscopic detection of specifically stained antigen (bacteria or viruses) in tissue sections using tagged antibodies, and polymerase chain reaction (PCR), which is a molecular technique performed directly on tissue or fecal samples for detection of the organism in question.

The Iowa State lab began using a broth dilution technique for obtaining antimicrobial susceptibility results about 18 years ago. This semi-automated computerized system provides quantitative information about the level of antimicrobial required to inhibit the growth of a particular organism in vitro (MIC or minimum inhibitory concentration) instead of a zone-size interpretation obtainable with the older Kirby Bauer disk diffusion technique. “We are able to purchase plates that are customized for the particular species group with which we are working,” says Hoffman. “This system has improved the quality of results, provided the option of rapid computer accessibility and increased our ability to monitor resistance of animal pathogens at the ISU VDL.”

Diagnosticians are wonderful teachers and most enjoy working one-on-one with veterinarians, adds Hoffman. “No question should go unanswered, and none should be considered insignificant. It is my opinion that each veterinarian working with a diagnostic lab should attempt to visit the lab, go to their Web site and become totally familiar with the faculty and staff expertise. A wonderful relationship with your diagnosticians, including the ability to ask questions about what samples to submit and how to submit them, generally results in an accurate diagnosis. Filling out submission forms properly and thoroughly also facilitates the process of diagnostic support.”

For more information

For information on diagnostic services and sampling, visit the Web site of the diagnostic laboratory you use.

For more information on shipping samples, see:

  • Shipping samples – know the regulations, Bovine Veterinarian, January 2002
  • Packing samples for shipment,  Bovine Veterinarian, March 2002

 

The best way to ship blood samples is to spin them down and ship the serum in Falcon tubes.

Sampling do's

Leann Siedlik offers these suggestions for collecting and sending samples for bacteriologic testing to the diagnostic laboratory.

  • Liquid samples should be placed in tubes or containers that can be tightly sealed and placed in a second Whirlpak® or sealable container for shipping.
  • Siedlik says her lab prefers getting larger tissue sections (i.e. lung) and suggests that the tissue be double-bagged in leak-proof bags and sealed tightly.
  • It is very important that the submission form be placed in a plastic bag and protected from the tissues so that it does not become blood-soaked. This seems like a simple request but there are many veterinarians who place the forms with the tissues.
  • Labeling the bags is very helpful, especially when the samples are from different animals or multiple clients.
  • Submission forms should be filled out to provide adequate demographics and background information.

Handling fad cases

Veterinarians are trained to watch for potential foreign animal diseases.

Randall Crom, DVM, director of Emergency Programs for USDA-APHIS Veterinary Services’ Emergency Management unit, says if veterinarians find themselves in a situation where they suspect they may be dealing with a foreign animal disease of some kind, they should contact their State Veterinarian or Area Veterinarian-in-Charge immediately. A decision can then be made, based on the specifics of the situation, as to the best way to proceed.

If, for some reason, a practitioner is unable to reach either of those offices, as a backup option they may call APHIS-VS Emergency Management at 800-940-6524.