Avibacterium paragallinarum FEATURES AND CULTURE

          Avibacterium paragallibarum  FEATURES AND BACTERIOLOGICAL CULTURE

    

             Carlos Rodríguez G      Biofarma Laboratories  (carlosrodriguezg25@gmail.com)    

Formerly known as Haemophilus gallinarum, Haemophilus paragallinarum and now Avibacterium paragallinarum Ap  is a gram negative bacteria, grows well in the egg yolk sac of embryos incubated at 5 or 6 days.

For culture need a broth containing at least 20% meat peptones, 5% meat extract, 5% dextrosa,. 5% sodium chloride and 2.5% sodium diphosphate, the culture also requires chicken serum 5% and an enzyme, nicotinamide adenine dinucle on reduced form DPN 5micrograms per ml of broth,.

As shown is a special medium that is not in the market and have to design.

Furthermore our culture must be done in a temperature reactor 37 degrees Celsius and maintains  at less 600 revolutions per minute RPM.

It should look a lot for bacterin production, but if it is isolated for the first time can be done in agar blood and Staphylococcus epidermidis, as mothership, the DPN enzyme excreted Staphylococcus colonies  is closest to the mothership, are larger, they are all as fine droplets of water this phenomenon we call satellitism and is very characteristic of the bacteria, in crops used instead of blood agar, brain heart infusion with chicken serum at 10% in these cases observed through a light source  you can see iridescence phenomenon, magenta toned

The isolates from the sinuses, are achieved in a cut in the form of a small triangle, find a clear and abundant mucus obtained a swab and is planted in the agar when the mothership placed dry, well seeded agar incubated in a wide mouth jar with a candle which burns to close the bottle depletes the oxygen content is thus achieved the crop to grow with a low amount oxygen, the cultures were obtained in less than 24 hours and are representative of a positive diagnosis of Avian Coryza.

Not just any insulation can make a bacterin. Making a bacterin effectively requires all three serotypes include A, B and C. The handling of these serotypes and conservation can explain it with an appendix scheme of how to treat each serotype to always have the same standard antigenicity.

As for my experience in growing Actinobacillus paragallinarum (Haemophilus paragallinarum), tests were made ​​with different means brand and did not find any effective and low price, I have used for over 20 years the following formula:

Primagen     20.0 g .. tissue culture medium

Meat extract            5.0 g. Difco

Sodium chloride       5.0 g.

Sodium diphosphate. 2.5 g
Dextrosa                   2.5 g.

1000 ml double distilled water.

This medium should be adjusted to a pH of 7.2

Sterilize at 120 degrees Celsius, a kilo of pressure for 20 minutes.

The chicken serum and beta Nicotinamida Dinuclotido salt disódica  a reduced form (NADP, coenzyme II) N 605 Sigma Chemical Co. sterilized by filtration sterility testing are made ​​and used as quickly as possible because it has enzymes reducing inhibitory potency.

Serum and NAD are added to the culture broth under safe sterility and if grown in a fermenter. Should be no more than 600 rpm, the culture has a generation time of 18 minutes

To get the chicken serum is purchased on the trail of  birds, 20 30 liters of  blood,  is placed in a colander made ​​expressly to filter clots and left for 24 hours at a temperature of 5 degrees Celsius (cold room).

The filtrate clarity fic to spin in a centrifuge made ​​for this purpose have baskets for a 500 ml bottle. for 30 minutes, then passed through a filter press cloth containing Pellyon and Decalite  (filter media), after two or three passes serum should be clear and bright with red wine-like appearance.

Forr serum sterilized, filtered by a 0.4 micron cartridge at 0.2, and then rated by a membrane filter of 0.4 and 0.2 microns in series, all Sartorius brand. Filters that get sterilized and ready for use.

There are other brands, but after several validation tests, the Sartorius brand proved to be the safest in terms of quality control.

If you do not have the Primagen peptone and meat extract, you can buy in a butcher Heart ground beef and ground beef.

200 grams of heart beef and per lt. 50 grams of ground beef, left in  1 lt. distilled water for 24 hours to make an infusion, at 24 hours boil for 20 minutes and stored in a freezer at 5 degrees Celsius for 18 hours.

After that time, the stock will have a thick layer of bait, which is removed, filtered with a blanket, and the filtrate is passed to a filter press, until the broth clear and bright like the beer.

The broth can be frozen or used immediately.

For use as a culture medium should add Sodium Chloride and diphosphate, adjusting the pH to 7.2, autoclaved,  20 kg. pressure, 20 minutes. 

    PRINCIPLES OF TRANSFORMATION 
      In Avibacterium paragallinarum (A-p)         

                                    

When still unknown intelligent molecule, which is how some geneticists call to ADN before 1928 many experiments were made ​​to kow the material that made ​​the cells replicate and inherit the characteristics of their progenitor, on this time Frederick Griffith used the information that some pneumococcal strains could mutate smooth to rough and back and made ​​a series of experiments with virulent and avirulent pneumococci.

He inoculated virulent type III pneumococci heat-killed a group of mice. Applied to another group type II pneumococci avirulent and a third group with  the two.pneumococci avirulent and virulent

The result was that the first two groups did not get sick, in the mice and the third group, killing all, showing that the genetic material of pneumococci had spent living dead type III to type II, the latter phenomenon was known first as Griffith effect and later Transformation.

There were other researchers who perfected these experiments by combining rough pneumococcal cells type II, type III smooth cells. These experiments demonstrated the passage of genetic material of living cells to the dead, this phenomenon is not exclusive of pneumococci are now known to occur between different bacteria,  Haemphilus, Pasteurella, Bacillus subtilis, Shigella etc.

So that taking advantage Avibacterium and Pasterella and have certain common characteristics, it’s  possible to get Avibacterium strain that not need the DPN and serum veside to retain their antigenicity and their pathogenicity, without having a Sequencer or other modern  genetic equipment .The important thing is to arm ourselves with a lot of patience and work hard to find what we seek.

We we’ll get for example, a vaccine of Pasteurella, CU strain, subject it to heat, checking that has been inactive or dead gives the mix a  one Avibacterium serotype.,  put in a medium such as agar Heart Infusion Brain and  without Serum and DPN, may be the poisibility that in no one culture tubes find a colonies, but if we're lucky we can find some colonies across the petri dish culture. Now comes the job of taking each colony and suspend it in a tube containing Infusion Brain, Heart broth media, the tubes that showing growth must be taken separately and inoculate 5 chicks six weeks old, it’s possible  in one culture find lesions coryza avian isolate of these are again Avibacterium, now we can test the patogenicity and antigenicity of the serotipe you work.

References

Principles of Genetics, Jhon Wiley 1988

Isolation And Identification Of Avian Pathogens Editorial commitee for The American Association of Avian Pathologist Texas A & University College Station

The Production And Use Of  Newcastle Disease Vaccines. W.H. Allan, J.E Lancaster and B. Tóth

Food and Agriculture Organization Of The United Nations FAO
Patrick J. Blackall  Personal comunication
MV. Carlos Rodríguez G. Labs. Biofarma, Personal Investigation

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