Bordetella bronchiseptica

Bacterium : Bordetella bronchiseptica

Classification : family of Alcaligenaceae.

Host species(ies) : dog, cat, pig, rodents, primates and humans

Infection / Associated Pathology : In dogs, Bordetella bronchiseptica belongs to the Kennel Cough complex.
In cats, Bordetella bronchiseptica may be involved in coryza syndrome.
In pigs, the bacterium is responsible for atrophic rhinitis.

Characteristic : Coccobacilla.

Transmission : direct contact (oral-nasal secretions).

Prevalence : asymptomatic carriage possible.
Mainly animals in groups and especially young dogs/cats that show more severe clinical signs.

Resistance in the environment : Low, sensitive to conventional disinfectants (bleach), dry heat, moist heat and cold.

Clinical signs :
Incubation for 3 to 10 days, then:
In dogs: cough, tracheobronchitis, rhinitis, bronchopneumonia in the broadest sense.
In cats: bronchopneumonia, nasal discharge, discharge from the eyes, cough, dyspnea.
In kittens: high mortality.

Prevention / Vaccination :
Vaccine: Yes (alive or killed).

Learn more about the diagnostic du virus de bordetella-bronchiseptica féline par PCR en temps réel.

Calicivirus (FCV)

Virus : Feline calicivirus or Feline Calicivirus (FCV)

Classification : family Caliciviridae

Host species(ies) : Cat and Cheetah

Infection/Associated Pathology : Feline Calicivirus

Characteristic : RNA virus that is very genetically unstable, hence the existence of many strains of Calicivirus of varying aggressiveness.

Transmission :

• direct contact between cats, through oronasal and conjunctival secretions
• Indirect contact possible due to resistance of the virus in the external environment

Prevalence : high prevalence, from 10 to 30% depending on the population, with possible asymptomatic carriage.

Resistance in the environment : persists for several weeks in the outdoor environment, especially on dry surfaces. Sensitive to concentrated bleach and quaternary ammoniums.

Clinical signs :

• Oral pathologies: stomatitis, gingivitis
• “Coryza” syndrome (frequent co-infection with feline herpesvirus, Mycoplasma felis and more rarely with Chlamydophila felis): respiratory signs (nasal discharge, eye discharge, etc.)
• Serious systemic disease: caused by hypervirulent strains leading to edema, polyarthritis, fever, skin ulcers, etc. and most often fatal.

Prevention / Vaccination :
Vaccine: Yes.

Learn more about diagnosing Feline calicivirus by real-time PCR.

Chlamydophila

Bacterium : Chlamydophila felis

Classification : family of Chlamydiaceae

Host species(ies) : cat (zoonotic risk not proven)

Infection/Associated Pathology : chlamydophilosis (formerly chlamydia).

Characteristic : obligate intracellular bacterium mainly of the conjunctival and nasal epithelia.

Transmission : direct contact (oculo-nasal secretions),
Probable transmission in utero

Prevalence : asymptomatic carriage possible in particular in animals living in or from these communities

Resistance in the environment : weak, but survival of a few days
in eye discharge at room temperature.

Clinical signs : more common in young cats less than 1 year of age.
Incubation for 2 to 5 days, then:

• “Coryza” syndrome (more or less in association with feline herpesvirus) with mainly ocular signs: conjunctivitis most often bilateral more or less mucopurulent, chemosis, blepharospasm, etc. and sometimes associated with respiratory signs: sneezing, rhinitis, etc.
• Abortion

Prevention / Vaccination :
Vaccine: Yes

Learn more about the diagnostic du virus de la chlamydia féline par PCR en temps réel.

Direct vs. Indirect Diagnosis

Laboratory diagnosis of infectious diseases involves two main types of techniques:

• so-called techniques Direct that allow you to search for the pathogen or part of it (antigen, genome)
• Techniques Indirect that highlight the Host response to infection (most often humoral or “serological” immune response).

The demonstration of an immune response in an infectious process, if it is easy to implement, can also sometimes cause problems of interpretation. This is particularly the case:

• when vaccination has been carried out for the suspected disease,
• when it comes to differentiating between a recent or older infection (evidence of an increase in antibody titer or “seroconversion”)
• in a young animal to which maternally derived antibodies have been transmitted,
  The development of a detectable immune response can be disrupted when the infection causes immunosuppression. The typical case is distemper in dogs.

In all these cases, it is interesting to use direct techniques to detect the infectious agent :

• cultivation,
• detection of the pathogen by microscopy,
• immunoassay for the detection of antigens...
• PCR or real-time PCR and other molecular biology techniques

The Direct and indirect diagnostic methods are complementary, but for the same disease, One or the other may be more interesting depending on the case (age, treatment, duration of evolution).

Molecular Diagnostics

A set of detection, quantification or typing methods targeting a nucleic acid (DNA or RNA). The Forms (and derived methods) is the most widely used molecular diagnostic method.

IVF

Virus : Feline Immunodeficiency Virus or Feline Immunodeficiency Virus (IVF)

Classification : Lentivirus of the family Retroviridae, RNA viruses

Host species(ies) : Domestic cats and wild felids

Infection/Associated Pathology : Feline immunodeficiency ( « page » you chat)

Characteristics : very genetically unstable virus

Transmission : Mainly through saliva (bites). Possible in utero and at birth.

Prevalence and asymptomatic carriage : The prevalence of FIV has decreased significantly in Europe over the last 20 years thanks to health and vaccination prophylaxis measures.
> Plus d’infos sur la prévalence des rétrovirus félins

Resistance in the environment : very low, on the order of a few minutes in the outdoor environment. Sensitive to all common disinfectants, including regular soap.

Clinical signs :
There are 3 stages in the evolution of the disease:

• Acute phase: this follows infection with the virus (fever, dejection, polyadenomegaly)
• Asymptomatic phase: the cat is a carrier of the virus but has few symptoms (gingivo-stomatitis, rhinitis, lymphadenopathy, weight loss, etc.). This phase can last for several years or even a lifetime.
• End-stage: The cat's immune system is exhausted.
  FIV is a virus that gradually weakens the cat's immune system; the clinical signs seen in infected cats are very often due to secondary diseases and not to FIV directly.

Prevention/Vaccination :
Vaccine: No
Neutering to prevent runaways and fights between cats.

Learn more about the diagnosis of feline immunodeficiency virus by real-time PCR.

Feline herpesvirus (FHV)

Virus : Feline herpesvirus or Feline Herpes Virus (FHV or FHV-1)

Classification : Family of Herpesviridae

Host species(ies) : Cat and wild felids

Infection/Associated Pathology : Feline viral rhinotracheitis

Characteristic : Genetically stable DNA viruses

Transmission :

• Direct contact between cats,
• Most likely transmission in utero

Prevalence/Carriage : Asymptomatic carriage possible with alternating phases of viral latency and reactivation. Reactivation of the virus can take place without causing clinical signs, which means that a cat can be shedding and therefore contagious even though it does not show any signs. The cat remains infected for life. The prevalence is around 40% in chronic or recurrent oculo-respiratory disorders.

Resistance in the environment :
Low, sensitive to heat, acids and most conventional disinfectants

Clinical signs :

• Ocular pathologies: conjunctivitis, keratitis, corneal sequestration
• “Coryza” syndrome (frequent co-infection with feline calicivirus, Mycoplasma felis and possibly Chlamydophila felis): respiratory signs (nasal discharge, eye discharge, sneezing, etc.)
• Dermatosis Skin
• Stillbirth kittens born to infected mothers, suspected role in certain reproductive disorders

Prevention / Vaccination :
Vaccine: Yes.

Learn more about diagnosingFeline herpesvirus by real-time PCR.

LIMS

It is the abbreviation of Taboratory Information Management System.
The Scanelis veterinary laboratory has deployed a computerized laboratory management system (LIMS) in order to automate the Full traceability of samples from registration to results and invoicing.

Feline Parvovirus

Virus : Feline Parvovirus or Feline panleukopenia virus or FPV (exceptionally CPV2, canine parvovirus).

Classification : family of Parvoviridae.

Host species(ies) : cat, wild cats, raccoon and ferret.

Infection/Associated Pathology : feline panleukopenia or feline parvovirus or typhus.

Characteristic : tropism for dividing cells: cells of the digestive tract, bone marrow, and lymphoid organs.

Transmission :
Direct contact: faeces mainly, weaker in saliva, urine and vomit.
Indirect Contact: Environment

Prevalence : asymptomatic carriage possible (common in young animals in a community)

Resistance in the environment : very resistant (several months in the outdoor environment).
Sensitive to liquid formalin and bleach associated with quaternary ammoniums.

Clinical signs :
Serious illness, especially in kittens between 6 and 14 weeks of age. Incubation period of 2 to 10 days.

• Superacute form: sudden death within 24 hours without clinical signs.
• Acute form: gastroenteritis, sometimes haemorrhagic, severe depression, hyperthermia, intense dehydration, leukopenia, sometimes neurological disorders.
• Cerebellar hypoplasia or retinal atrophy of the kitten when the mother is exposed to the virus during gestation.

Prevention / Vaccination :
Vaccine: Yes.
Prevention: sanitary measures on farms, shelters.

Learn more about the diagnosis of panleukopenia by real-time PCR.

Forms

Discovered in 1986 by Kary Mullis (Nobel Prize in Chemistry in 1993), PCR or Polymerase Chain Reaction is a Method for direct detection of the genome of infectious or parasitic agents by enzymatic amplification of a part of it. This molecular biology tool is very specific and makes it possible to reproducibly detect very small quantities of pathogens in samples of various kinds.

In genetics, PCR is a first step in the study of a given genomic region. It makes it possible to target this region to look for polymorphisms associated with a disease or a particular susceptibility to certain infectious agents or drugs.

The principle of PCR

Two primers (oligonucleotides of approximately 20 bases) specific for the fragment to be searched for and located at each end of the target are used as “seeking heads”.
Well chosen, these primers are unable to hybridize efficiently elsewhere than on the target fragment, which explains the specificity of PCR.
Once the target is found, a heat-stable enzyme (Taq polymerase) synthesizes new DNA fragments similar to those of the original sequence present in the sample.
These cycles of primer hybridization and DNA polymerization are repeated at least 30 to 50 times, resulting in an exponential multiplication of the original target sequence.
This amplification explains the Sensitivity of PCR techniques (or rather their very low detection limits: the presence of a few copies of the viral genome gives rise to several billion identical fragments in two hours).

You can also check out our animation presenting PCR and real-time PCR techniques.

The advantages of the...

PCR is a Highly efficient means of direct diagnosis of infectious or parasitic diseases, especially in the early stages of infection or when vaccination or the presence of maternal antibodies are likely to interfere with serological diagnosis (indirect diagnosis).
The Speed of results (in Scanelis, D+1 to D+2) can be a decisive advantage, especially if the suspected disease is a zoonosis (leptospirosis), if the rapid implementation of sanitary measures is necessary in a farm (distemper, parvovirus) or if the treatment must be adapted to the pathogen present (Piroplasmosis in Equidae).
The shipment of specimens does not require any special precautions and it is possible to search for several pathogens simultaneously in the same sample.

And its limitations...

The limitations of PCR are the difficulties in designing, implementing, and interpreting the tests.
In design, it is not enough to choose two specific primers to develop a relevant PCR test. Many factors influence the actual performance of the technique, such as:

• the Choice of direct debit (which must contain the infectious agent),
• the Sample processing techniques prior to PCR (adapted to each type of sampling),
• Optimization and validation of the method (choice of reagents, equipment and reaction conditions, inclusion of appropriate controls: Is the detection threshold checked on each series?detection of PCR inhibitors).

The The choice of technology is decisive. For example, it is unrealistic to hope to diagnose parvovirus in a vaccinated puppy or FIP in a cat if quantitative PCR (real-time PCR) is not used.

During implementation, a certain number of pitfalls must be avoided (contamination of samples in the laboratory, presence of inhibitors, etc.). It is therefore necessary to Turn to a specialized and experienced laboratory in these technologies.
In addition The interest of the result obtained is closely linked to the quality and choice of samples : sampling methods adapted to the pathology, choice of sampling according to clinical evolution, richness of cell sampling for swabs, cytobrushes, etc.
For the interpretation of the results, it is also necessary to Know the percentage of asymptomatic animals with the test used.

Real-time PCR

Evolution of PCR, the real-time PCR technique, mastered by Scanelis since 2000, makes it possible to detect genetic sequences characteristic of the agent to be searched for. All tests developed and marketed by Scanelis use the real-time PCR (quantitative PCR) technique.

Principle of real-time PCR

As in PCR, two primers (oligonucleotides of approximately 20 bases) specific for the fragment to be searched for and located at each end of the target are used as “seeking heads”.
A labeled probe, also specific to the target sequence and internal to the two primers, is added to the reaction medium. The amplified fragments are highlighted as the reaction cycles progress.
It is also possible to use a DNA intercalant instead of the probe to reveal the amplification, but these methods, which are less expensive in terms of reagents, are also less specific (delicate or even impossible typing).

You can consult our “Real-time PCR” animation.

Major advantages over conventional PCR methods

• Quantitative analysis results if needed
• Stump typing using specific fluorescent probes (e.g. vaccine strains/field strains)
• Reproducibility
• Sensitivity very important
• Outcome security (No risk of contamination and false positives due to lack of electrophoresis analysis)
• Automated analysis

The development of quantitative PCR/real-time PCR tests requires real biomolecular expertise.

quantitative PCR

This term is often used to talk about real-time PCR.

Quantitative PCR allows the evaluation of viral or parasitic loads. It is a technique that has many advantages compared to conventional techniques .

Quantitative PCR allows:

• pertaining to monitor the effectiveness of a treatment ;
• pertaining to distinguish a simple carriage from an actual infection (one of the major problems in the interpretation of PCR results, which is a very sensitive method);
• pertaining to distinguish between animals vaccinated with an attenuated strain and those actually infected (canine parvovirus, distemper, etc.). This technology also offers the possibility, in some cases, to distinguish between pathogenic and vaccine strains.

RT-PCR

When the genome to be searched for is an RNA (RNA virus), a preliminary step to the PCR reaction makes it possible to make a copy of the target RNA fragment into DNA using an enzyme isolated from retroviruses: reverse transcriptase. This is called RT-PCR.

A few examples of RNA viruses of veterinary interest :

• Distemper virus
• Feline Coronavirus and Canine Coronavirus
• Feline calicivirus
• Feline retroviruses: FeLV and IVF
• Pestivirus bovin
• Rabbit Viral Hemorrhagic Disease (VHDV) Virus

Detection threshold

The detection limit for a test is the smallest amount of target sequence that can be detected by analysis. In practice, the detection limit is expressed at Scanelis as a number of target sequence copies (on the genome of the pathogen being sought).

For each test developed by Scanelis, this threshold is determined in accordance with the recommendations of the European Pharmacopoeia and the NF U47-600-2 standard and verified on each series of analyses.
A statistical analysis on a large number of standard points of several different dilution ranges (at least 24 determinations per quantity) is therefore performed to determine the detection limit at 95% (Probit analysis). The detection limit provided (close to 100% under the conditions under which the tests are performed) is equal to the 95% detection limit multiplied by a factor of 3. Positive standards corresponding to this threshold are integrated into all series of analyses, which ensures the reproducibility of the results. For each test, the detection limit is indicated on the Scanelis PCR Test Specification Document.

Quantification threshold

The quantification threshold is the smallest amount of target sequence (in number of copies) that can be accurately quantified by the real-time PCR method (which makes it possible to discriminate quantities varying by a factor of 2).

For each test developed by Scanelis, the linearity domain of PCR is determined according to the recommendations of XP U47-600-2 and the lower bound of this domain is the limit of quantification.

In practice, a positive result “below the quantification threshold” means that a small amount of the target (virus, bacteria, etc.) has been detected with certainty in the sample but the amount is too small to be accurately measured.