3. DIAGNOSIS: Let's solve the brain teaser

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3. DIAGNOSIS: Let's solve the brain teaser


Article written by:

Jesús Salinas, Nieves Ortega, María Rosa Caro
Department of Animal Health, Faculty of Veterinary Medicine, University of Murcia. 30100 Murcia, Spain.


As is the case with all infectious abortive diseases, early diagnosis is essential to establish hygiene-sanitary measures and the treatments necessary to limit the clinical and economic consequences of the infection on the affected flock.


In addition, it will allow for the establishment of the appropriate prophylactic or vaccinal means to avoid an increase of abortions in the following years.


Figure 2: Diagram showing the main methods used in the laboratory diagnosis of OEA depending on
the samples collected after an outbreak of abortions.

 

However, given that the clinical and epidemiological signs are not specific, in the majority of cases it is essential to resort to laboratory diagnosis to confirm the cases of abortion due to chlamydia (Figure 2).
 

Clinical signs and epidemiological data


The clinical signs that show a flock affected by OEA are not sufficiently clear to reach a certain diagnosis of the disease.


There are other abortive diseases in small ruminants that can have similar symptoms, such as Q fever, brucellosis, paratyphoid abortion or toxoplasmosis, which is why the only way of issuing a certain diagnosis is laboratory confirmation.
 

Laboratory Diagnosis: The only way to confirm


The reasons mentioned above make it necessary to carry out a diagnosis in the laboratory, which may be direct from products of the abortion, or indirect from serum samples.


1. Choosing of samples


For the conduct of a direct diagnosis, the sample of choice is the placenta.


Blood samples from animals recently aborted for indirect diagnosis

 

Several cotyledons must be collected and placed in sterile plastic bags or fixed in formalin, depending on the analyses requested (bacteriological, molecular or immunocytochemical).
 

Placenta is the ideal sample for direct diagnosis techniques


If the placenta is not available, samples taken from the foetus (spleen, liver, contents of the abomasum or lungs) can be analysed, even if in these cases it is appropriate to resort to taking vaginal swabs after the abortion, since the excretion of the pathogenic agent in secretions is very high for several days or weeks.

 

Direct diagnosis: vaginal swabs taken within first week after abortion

 


On the other hand, indirect diagnosis is performed on serum samples.


In this case, it is advisable to take at least 10 samples from different animals that have aborted within the same flock. Given that the level of antibodies can reduce quickly, samples must be taken within 8 weeks after the maximum peak of abortions in the flock.
 

Serum samples must be taken from animals within 8 weeks after the abortion 


In the case of working with individual sera, from animals that have aborted, we must take two blood samples within 15-20 days (matched sera), in order to check if there has been seroconversion.
 

2. Direct diagnosis


This is a set of techniques intended for detecting the infectious agent on the pathological samples, which includes the following methods:

Figure 3: Direct diagnosis of OEA. Detection of C. abortus by means of:
A. Stamp’s stain. B. May-Grünwald-Giemsa stain. C. Immunohistochemistry on an histological section D. Immunofluorescence. 

 

  • Bacterioscopic examination: This is an analysis that enables C. abortus to be demonstrated in non-specialist laboratories. These analyses include those traditional differential stains, such as Stamp’s (Figure 3A) or May-Grünwald-Giemsa (Figure 3B), or more recently direct immunofluorescence (Figure 3D) or immunoenzymatic techniques (Figure 3C) for antigen detection, which are more specific and sensitive if monoclonal antibodies are used. These techniques are performed on cotyledon or vaginal swabs, and are less sensitive when carried out from foetal organs. The cotyledon samples may also be fixed in formalin and included in paraffin for the conduct of immunocytochemical techniques on histological sections.

 

  • Culture: Given the obligate intracellular microorganism character, the use of living cells is required for its culture. The first method used to cultivate chlamydia used the yolk sacks of chicken embryos, which continue to be used routinely in many laboratories. However, the development of the culture methods on cellular lines has moved considerably to embryonic eggs. The most used sensitive cell lines are the McCoy, Vero, HeLa 229 or fibroblast L-cells.

 

  • Molecular techniques: The possibilities of rapid and precise detection of chlamydia on clinical samples have greatly improved since the introduction of molecular methods, particularly gene amplification techniques (PCR), which can be conducted on the same samples, both fresh and frozen, or even on samples fixed in formalin. Unlike conventional PCR, real-time PCR allows for the quantification of the quantity of chlamydia there is in the sample.

 

Therefore, PCR is a rapid and precise direct technique

 


3. Indirect or serological diagnosis


The serological diagnosis of OEA is complex due to many factors, but especially due to the latent nature of the infection, which means that seroconversion does not occur until after the abortion.


Furthermore, ruminants are also often infected by C. pecorum, in many cases sub-clinically, but inducing the production of low, but detectable, quantities of antibodies.


For these reasons, this serological diagnosis must imperatively be a group or flock diagnosis.


It is considered that the flock is affected by a latent (or recently vaccinated) infection when we see rates of antibodies equal to or slightly higher than the positivity threshold of the techniques used, considering a flock supposedly exempt when all the serums studied have provided results lower than the established threshold.


The most used methods are the following:
 

  • Complement Fixation Test (CFT): For over 50 years, CFT has been the most used technique for the serological diagnosis of OEA, and is also recommended by the World Organisation for Animal Health (OIE). However, CFT lacks specificity due to the fact that the antigen used mainly consists of lipopolysaccharide (LPS), which is common to all species of the Chlamydiaceae family, making cross-reactions with the antibodies produced against C. pecorum common.

 

  • ELISA-type immunoenzymatic techniques: This is the most used technique at present. It is essential to use commercial kits that use specific recombinant antigens of C. abortus related to the 80-90 kDa POMP-based ELISA, ruling out those that use complete elemental bodies or LPS, since they can give rise to cross-reactions with the responses induced by C. pecorum.
     

The indirect technique of choice is ELISA 

 

Control & Prevention


There are three types of measures in the prevention and control of OEA:

  • General measures related to management, similar to those of any abortive process.
     
  • Treatment with antibiotics, such as tetracyclines, after an outbreak of abortions.
     
  • Vaccinal prophylaxis, with two types of vaccines available at present; attenuated live vaccines and inactivated vaccines.

 

 

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REFERENCES: 
 

Due to the extensive bibliography used in the writing of this article, the references have not been included in the text. If the reader would like further information about any aspect of this monograph, please direct your questions to the authors at the following email address: jsalinas@um.es

 


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