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Accurate Mycoplasma hyopneumoniae diagnosis challenging but essential

02 November 2017

Poultry Health Today

The diagnosis of Mycoplasma hyopneumoniae (M. hyo) infection remains challenging but can be made with confidence when a systematic approach is applied and the clinical picture aligns with laboratory tests, says Kent J. Schwartz, DVM, clinical professor at Iowa State University.

Even when pigs present with the hacking cough and labored breathing that points to M. hyo-induced enzootic pneumonia, the diagnosis needs to be confirmed since the signs of disease might be due to other causes, Schwartz told Pig Health Today.

Diagnosing M. hyo can be especially difficult when M. hyo colonizes the cilia of the respiratory tract. Weeks or months can go by before clinical signs appear. Meanwhile, feed conversion and average daily gain can suffer. In these cases, the clinical expression of M. hyo depends on many factors such as pig susceptibility, the M. hyo bacterial load and strain virulence as well as the presence of exacerbating bacterial and viral co-infections, he says.

Start with observation

The systematic approach Schwartz advises begins with clinical observation. “Simply observing pigs can help determine if M. hyo infection is present, but to be accurate, pigs should be routinely examined in their environment to identify illnesses, abnormalities and risk factors.”

Clinical signs of M. hyo infection tend to be seen late during the grower or finisher period. “That hacking cough that most pork producers can recognize is most likely to occur during exercise at the start of the day,” he says.

Fever generally isn’t seen with M. hyo infection alone, he says, and feed consumption is only modestly decreased in contrast to outbreaks of swine influenza or porcine reproductive and respiratory syndrome (PRRS). M. hyo-associated clinical disease is generally self-limiting unless pigs are immunologically naïve or they have complicating infections or abnormal environmental stresses.
The situation worsens when co-infections are present and porcine respiratory disease complex (PRDC) emerges as a complication of M. hyo infection. This is when mortality can dramatically escalate, Schwartz says.

Common virual co-infections in with PRDC outbreaks include PRRS virus, influenza A virus and porcine circovirus type 2. Common bacterial co-infections include Pasteurella multocida, Streptococcus suis, Haemophilus parasuis and Actinobacillus suis, the veterinarian continues.

Look for gross lesions

Clinical observation should be followed by necropsy to look for gross lesions characteristic of M. hyo infection — or other pathogens. “This is where you ask ‘What could be causing this disease, this lesion?’” Schwartz says.

M. hyo-associated pneumonia will cause grossly visible lesions in the lungs, usually with consolidation in the lower portions of the front and middle lobes. The amount of lung involved can vary and may exceed 10% of total lung volume in uncomplicated cases. However, lesions are quite variable between individual pigs, ranging from barely present to 30% of lung volume, he says. If more lung volume is affected, other bacterial contributors are likely involved, he says.

Histopathological evaluation

Histopathological evaluation is next on Schwartz’ list for the diagnosis of M. hyo. Microscopic examination of lung lesions is aimed at identifying tissue changes compatible with M. hyo involvement. It can also help rule out other respiratory pathogens.

Histopathology is performed on formalin-fixed lung sections, preferably from affected, euthanized pigs, since pigs that succumb naturally often have confounding co-infections, he says.

“Lung sections that are approximately 1x3x3 cm — the size of a matchbook — should be collected from every portion of lung that looks or feels different,” Schwartz says. The sections should include transitional (sections across both normal and abnormal) portions as well as visible airways. Usually, this will be from areas from higher portions of lung nearer larger airways rather than just the consolidated tips of various lobes.

M. hyo-specific tests

Schwartz recommends M. hyo-specific laboratory tests after assessment of clinical signs, gross lesions and histopathology since asymptomatic carriers of M. hyo are common.

Culturing for M. hyo will confirm the diagnosis, but it’s seldom used because it’s such a tedious, time-consuming and expensive process, he says.

Schwartz cautions that laboratories often differ in the repertoire of tests they offer as well as in the methodology they use. For these reasons, he advises against comparing numerical results from different laboratories.

The test of choice for determining if M. hyo is present in a sample or specimen is polymerase chain reaction (PCR). There are variations in PCR tests depending on the laboratory used — for instance, real-time PCR or “nested” PCR — but for all of them, the most important factor in obtaining an accurate test result is sampling technique and quality, he says.

Fresh samples should be transported on ice if shipping time to a laboratory is < 48 hours, but if shipping time is > 48 hours, then freezing the samples for PCR testing would be preferred.

Sample types can vary and are often influenced by factors such as collection-technique expertise, test availability or transportation logistics. Schwartz cites the preferred PCR sample types in the order they are most likely to detect M. hyo:

  • Bronchoalveolar lavage is considered the gold standard. Samples are easily obtained at necropsy and can be performed antemortem.
  • Airway swabs are also easily obtained at necropsy and can be obtained antemortem.
  • Affected lung tissue is yet another easy type of sample to obtain at necropsy. Go for samples proximal to consolidated tissue. Airways close to the hilus are most likely to harbor pathogens involved in pneumonia. Resist the temptation to submit only firm distal lobes. Fresh lung samples should be golf-ball sized and should represent each portion of the lung that looks or feels different.
  • Deep nasopharyngeal swabs require animal restraint and an appropriate swab design.
  • Tonsil scraping requires animal restraint, a speculum and a long-handled retrieval instrument.
  • Deep nasal swabs require animal restraint. They can yield positive results in some symptomatic pigs. Oral fluids are easy to obtain and are useful for detection of hyo in symptomatic populations but are poor samples for determining if asymptomatic populations are subclinically infected with M. hyo.

Is M. hyo causing disease?

Even if M. hyo is found to be present, it doesn’t prove it’s the cause of disease. Tests that can answer that question are immunohistochemistry (IHC), fluorescent antibody techniques (FAT) and in situ hybridization (ISH) because they enable visualization of the specific organism within a typical lesion, Schwartz says.

All three techniques are highly specific for M. hyo, but they are inherently less sensitive than PCR because for M. hyo to be detected, they require a relatively large quantity of the pathogen to be present. The accuracy of these tests is highly dependent on sample quality and stage of disease, he emphasizes.

FAT is performed on fresh tissue, whereas IHC and ISH are performed on formalin-fixed sections taken from the same areas as FAT, Schwartz explains. He recommends submitting fresh and formalin-fixed samples from an animal in the early stage of acute clinical disease without evidence of substantial secondary bacterial involvement.

“Select a suitable portion of the gross lesion in lung. Slices of affected lung 1 to 2 cm thick should be obtained along with a transition area and unaffected lung; they should contain visible small airways,” he continues. The samples should be preserved by rapidly chilling for FAT testing or by prompt immersion in formalin for IHC and ISH (and histopathology). Never freeze samples for FAT, IHC, ISH, or histopathology.

Antibody detection

Serology performed with a commercial ELISA test is the most common and most economical way to determine herd-infection status. It can be useful for detecting M. hyo exposure in nonclinical herds but has poor test sensitivity. In other words, it may fail to identify infected, asymptomatic carrier animals, Schwartz says.

“Seroconversion doesn’t occur for some weeks after colonization and then only sporadically in colonized pigs. However, if you find a positive animal on serology, you should be concerned the population is colonized, and this is especially true when the herd hasn’t been vaccinated against M. hyo,” he says.

Wrapping it up

“Once you collect all your information using this systematic process, analyze, think and incorporate valid and relevant scientific literature into your conclusions,” Schwartz says.

“Ask if it all makes sense. Ask if the clinical findings align with gross lesions and M. hyo-specific tests. If the conclusion is that M. hyo is present and the cause of disease, then you can proceed with development of a treatment and control strategy,” he says.

More detailed information from Dr. Schwartz on gross lesion findings and diagnostic test interpretation are available in A Contemporary Review of Mycoplasma Hyopneumoniae Control Strategies.



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