. Amantadine Viricide effectiveness against Avian Influenza Serotype H5N2.

      Amantadine Hydrochloride Viricide Effectiveness against Avian Influenza             

        Serotype H5N2.

                              Carlos Rodríguez G Biofarma Laboratories  N.L. México October 2003

Summary.

Isolation of avian influenza A Serotype H5N2 in commercial lay hens in Northeastern Mexico, showed low pathogenicity, we studied the effect of Amantadine Hydrochloride viricidal to treat the disease, which in some cases is only manifested by positive serology. ( 5)

Amantadine Hydrochloride has been used as an antiviral against avian influenza (AI) since 1966, is effective in blocking human infections in birds and mammals, this drug was used in influenza outbreaks in turkeys, with relative success, until the treatment was abandoned when it was discovered that could generate amantadine resistant strains (3).

In this research, we try to demonstrate the viricidal effect of amantadine with serotype isolated in Northeastern Mexico and propose a treatment, according to  Webster, Kawaoka and Bean.(5). Taking the idea outbreaks with H5N2 serotype isolated showed very low pathogenicity and that some were detected only by positive serology unnoticed clinically

Materials and Methods

To demonstrate the effect viricidal Amantadine Hydrochloride; embryos were used 10 days of incubation from Leghorn hens, IA free flock.

To test the viricidal effect in birds reared two lots of 30 chicks Cobbs-Cobbs, well insulated and separate batches to which were given a hemagglutination inhibition (HI) test to demonstrate that they were free of antibodies to IA.

The positive serum HI tests was provided by the Laboratory of the Mexico-American Commission for the Eradication of FMD and other exotic diseases (CPA)

Amantadine is sold in Mexico as Hydrochloride USP chemically pure grade, but it was impossible to obtain a sample, we used Fluviatol Boehringer Ingelheim Laboratories SA de CV This for humans medicine contains 50 mg. Amantadine per tablet.

Procedure:

Batch A.For demonstrate viricidal effect in chick embryos, an inoculum was prepared containing allantoic fluid with AI virus with a titer of 10-7 Nutrient Broth with Penicillin-Streptomycin and Amantadine 5mg/ml of  this mixture 10 embryos inoculated via the allantoic, 0.1 ml per embryo (1).

Batch B or control group contained the AI virus with the same title and nutrient broth containing Penicillin-Streptomycin, 0.1 ml was inoculated embryos. (1)

Both batches were incubated for three days and canding twice a day.

The results are shown below:

Treatment  Embryos  Hours Embryo Mortality Agglutination test with GR

Batch          10              56        0/10                        +0 / 10

Batch          10              56        0/10                       +10 / 10

Note: GR are chicken red blood cells 5%

Checked the viricidal effect of Amantadine, in chick embryos, we designed a test in broilers reared Cobbs Cobbs-complete isolation, at 4 weeks the HI test showed they were free of antibodies to AI ..

Both batches  A and B with the same number of chickens were inoculated via eye drop containing allantoic fluid IA with a  10-7 titration

Batch A received Amantadine 50 mg per liter in drinking water  and food for 15 days.

Batch B received water  and food only during the same period.

Results

Batch A, No signs or symptoms of IA, during the trial period.

Batch B, Some hemilateral inflammation of the sinuses bilaterally without mucus, some rattling tracheobronchial, depression, low feed intake, these symptoms were observed for 5 days, then the chickens fully restored, the test was continued for 15 days.

Results birds  sinus inflammation  Respiratory sintoms  Mortality    IH (1)

A         30       0/30                                           0/30        +0/30

B          30     22/30                                          16/30      + 30/30

Note, IH hemagglutination inhibition test 

In both batches 5 samples were taken randomly from tracheal swabs within 10 days of the test, were suspended in Nutrient Broth with 100 micrograms per ml Gentamicin, Penicillin 1000 units per ml and Amphotericin B 5 micrograms per ml. Standardized techniques for virus isolation (1) is 10 embryos in 10 to 11 days of incubation. Each sample contained the antibiotic Nutrient Broth swabs, 0.1ml of inoculum per embryo, incubated and  candling twice a day and discard embryos that die during the first 24 hours for three days trial.

Results: HA

Batch  A +0 / 10

Batch  B +10 / 10

The isolate was identified by agglutination test with 5%  G.R HA (1)

Discussion and Conclusions.

The Amantadine Clohydrate proved effectiveness against IA in chick embryos and in broilers 4 weeks old

Moreover, the AI on farms where they became the isolates, showed few clinical signs, rales , low feed intake, mortality increased by 1% and in five days the birds began to recover. Some of these symptoms are replicated in the inoculated animals.

Amantadine according to the literature, acts against the neuraminidase of the virus (4) and functions as viricidal.

Amantadine hydrochloride is a water-soluble drug very low cost compared to Oseltamivir and Paramivir of human use and could be used in cases where the avian influenza strain is of low pathogenicity and proposing simultaneous vaccination with the same serotype, this would avoid the dissemination of the virus that gives positive serology, with few symptoms and economic losses.

REFERENCES

1. - Beard, CW Avian influenza: Isolation and identification of avian pathogens, ^2nd Ed S: BB. Hitchner, C: H: Domermuth, H: G: Purchase and J: E: ED Williams. Am Assoc. OF Avian Pathologist, Colege Station Tex. 66-69, 1980

2.- Beard, CW, Brugh M and Webster R: G: Emergence of Amantadine-Resistant H5N2 Avian Influenza Virus During a Simulated layer tratment Flock Program. Avian Diseases 31:533-537 1987

3.-Hay, A: J: M: C: Wainright Patricia O. Purdue L. Michael, Max and Beard W. Brugh Charles. Amantadine Resistense 5 Strains Among Hemagglutinin Subtype Avian Influenza Virus OF. Avian Diseases 35:31-39, 1991.

4.-There Wolstonhome AJ, JJ Skehek and M: H: Smith. The Molecular Nasis antiinfluenza Of The Specific Action of Amantadine HCL E: M: BO (Eur.Mol.Biol. Organ J. 4:3021-3024. 1985

5. - Webster, RG Y. Kawaoka, WJ Bean, CW Beard and M. Brugh. Chemotherapy and Vaccination: A possible strategy for controlling of highly virulent influenza virus. J. Viol. 55:174-176 1985

Rodriguez G.C. MédicoVeterinario Zootecnista Veterinary Faculty, (UNAM). Industrial Microbiology Master incomplete Faculty of Chemistry (UANL) Member of the Mexican Association of Microbiology. Former member of The American Association of Avian Pathologists. Biofarma Labs.