Int J High Dilution Res 2011; 10(37): 362-368

Original Article

In vitro behavior of Mycoplasmagallisepticum

live-type nosode

Môsar Lemos1, Elmiro Rosendo do Nascimento1, Maria Lucia Barreto1,

Virginia Leo de Almeida Pereira1, Cátia Cardoso da Silva1,
Jenif Braga de Souza2, Sabrina da Silva Barros2,
Leandro dos Santos Machado1, Carlos Augusto de Martino Campos1

[1] Veterinary Physician, Fluminense Federal University (UFF), Brasil

[2] Veterinary Physician, Center of Animal Experimentation, IOC/FIOCRUZ, Brazil;

ABSTRACT

As a step of a doctoral research project, in this study a live-type nosode was prepared from
microorganism Mycoplasmagallisepticum strain R (ATCC 93-08/19610) according to Costa model
and the rules by Brazilian Homeopathic Pharmacopoeia. Live nosode was tested in vitro to assess
safety when used to immunize domestic fowl (Gallus gallus) against infection by this
microorganism and to investigate its behavior under laboratory conditions. M. gallisepticum was
not shown to grow in fluid (broth) and solid (plate) modified Frey medium with dilutions 11d, 12d,
20d and 30d. Inhibition halos about 2.0 mm were observed around paper disks impregnated with
live-type nosode in microorganism-sown Petri dishes, whereas disks impregnated with
conventional antibiotic oxytetracycline exhibited 8.0 mm inhibition halos. Protein assessment by
Folin-Lowry method showed protein absence in dilutions 12d and 30d and neither microbial DNA
traces were found in PCR assay in dilutions 12d, 20d and 30d.
Key-words: Biotherapy; Chickens; Live nosode; Mycoplasmagallisepticum

Introduction

According to Brazilian Homeopathic Pharmacopoeia, biotherapy or nosodes are “medicinal preparations

intended for homeopathic use obtained from chemically indefinite biological products: secretions, excretions,
tissues and organs, be they pathological or not, microbial products and allergens”. [1] They can be used for
prophylaxis purposes, since they correspond to the etiologic agent of some disease used as starting-substance.
[2,3] Nosodes are traditionally considered homeopathic medicines because they are prepared according to
homeopathic pharmacotechnics using a disease etiologic agent as starting substance; an alternative use to
protect and treat animals against various diseases has been tested for several years. [4,5]

The expression live nosode is used to distinguish between nosodes prepared from dead cultures and used in
clinical practice according to their pathogenetic effects, and nosodes prepared from non-inactivated (live)
cultures; the original model for the latter was live culture of Neisseria meningitidis. [6] According to Costa,
“dynamized living nosode elicits production of immunizing and blocking antibodies, thus certainly causing
exacerbation of natural immunity, and immunological resistance, surveillance and homeostasis”. [3]

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Among microorganisms affecting commercial production of chicken, genus Mycoplasma is one of the major
causes of aviculture harm; this taxon is composed by small prokaryote lacking cell wall organisms.
[7]Mycoplasma infections may treated by means of antimicrobial agents in order to improve the overall state
of fowl, reduce or eliminate clinical symptoms, reduce or inhibit dissemination of disease and vertical
transmission of the etiologic agent, and reduce the mortality rate and the severity of damage caused by
pathogenic strains, [8]; however, they may leave residues in treated fowl.

Residue is defined as any compound present in foodstuffs resulting from the metabolism or degradation of
veterinary drugs, antimicrobial agents, food additives, heavy metals and pesticides, among others, which pose
a risk for dietary safety when their levels surpass the maximum levels recommended by the National Agency
of Sanitary Surveillance – ANVISA. [9]

Wilhelm Lux was a veterinarian doctor, professor at Leipzig Veterinary School and contemporary of Samuel
Hahnemann, moreover, he was one among the first veterinary doctors to employ biotherapy agents. Lux was
called to treat an epidemic of hematic carbuncle; since he lacked homeopathic medicines matching the clinical
image of disease, he treated this epidemic with dynamized blood of an ill bovine and obtained positive results.
Later on he solved and epidemic of glanders in Hungary by diluting and agitating nasal discharge of an ill
horse. In this way, Lux established the basis of biotherapy. [10,11]

Materials and methods

Preparation of live nosode

Live nosodeMycoplasmagallisepticum (strain R ATCC 93-08/19610) – MGR –was prepared in laminar flow
using amber-hued vials previously sterilized in autoclave (121ºC/30 minutes) according to the technique
recommended for live nosodes. [2] Live microorganism culture in suspension containing 3 billion cells per mL
was used as starting-substance; serial dilutions up to 30d were performed according to the guidelines of
Brazilian Homeopathic Pharmacopoeia. One mL of M. gallisepticum culture was diluted in 10.0 mL normal
saline, then serial dilution and agitation was performed until 30d. Up to 10d, solvent used was normal saline,
11d was prepared with 5% hydro-alcoholic solution, whereas for higher preparations solvent used was alcohol
60ºGL.

In vitro inhibition of Mycoplasma growth by live nosode

To establish the ability of the live nosode to inhibit M. gallisepticum growth in vitro, paper disks were
impregnated with biotherapy in dilutions 12d and 30d and oxytetracycline aseptically in laminar flow and
placed at the center of Petri dishes sown with M. gallisepticum, which were incubated at 37ºC for 21 days or
until characteristic colonies appeared. This test was replicated five times.

In vitro test of live nosode innocuousness

Dilutions 1d, 10d, 11d, 12d, 20d, 30d of biotherapy were tested for M. gallisepticum growth by means of
culture in fluid and solid Frey medium. After inoculation, samples were incubated at 37º C for 21 days; they
were rated negative after this time elapsed.

Protein dosage

Protein was dosed by means of Folin-Lowry method [12] in mother solution and dilutions 1d, 12d and 30d.

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Polymerase chain reaction (PCR)

Dilutions 1d, 5d, 10d, 11d, 12d, 20d, 30d and mother solution (MS) were tested for presence of M.
gallisepticum DNA fragments; DNA was extracted from samples by means of phenol/chloroform method
following Sambrook et al. [13] After DNA extraction, each sample was concentrated in ethylic alcohol
according to Zeugin & Hartley, [14] then centrifuged, the sediment was suspended in Tris-EDTA buffer pH
8.0 and stored at 20º C. DNA amplification reaction was performed in thermocycler for 40 cycles using
primers B1 5’ CGT GGA TAT CTT TAG TTC CAG CTG C 3’ and B2 5’ GTA GCA AGT TAT AAT TTC CAG
GCA T 3´ (481 pb), to detect M. gallisepticum. Amplified products were added tracking buffer and subjected to
electrophoresis in 1.5% agarose gel submerged in TBE O.5X buffer. [13] After electrophoresis, gel was stained
with ethidium bromide, results were visualized in a transilluminator under ultraviolet light and
photographed.

Results and discussion

In vitro test of live nosode innocuousness

No growth of M. gallisepticum was observed in cultures with dilutions 11d, 12d, 20d and 30d, whereas
dilutions 1d and 10d exhibited microorganism growth. This agrees with Costa, [2] who stated that live nosode
is innocuous from dilution 12d onwards. Inactivation of MGR is due to the change of solvent from normal
saline to alcohol 60º GL (Figure 1, Table 1).

Table 1: Growth of MGR 19610 colonies in several dilutions

Dilution/Day 1st day 3rd day 5th day 12th day 17th day 21st day
1d +
10d - +
11d - - - - - -
12d - - - - - -
20d - - - - - -
30d - - - - - -

Figure 1: Growth of MGR 19610 living nosode

in Frey medium

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These results agree with Costa, [2] who stated that dilution 12d is absolutely innocuous as to the possibility of
causing contamination, therefore, the proposed live nosode in dilution 30d is safe for use.

Results also agree with Siqueira, [15] who assessed cell alterations induced by a new live-type nosode on lines
MDCK e j774.g8 and concluded that hemagglutination was able to detect virus titers < 2 HAU/25 μl in
supernatant of fertilized eggs inoculated with Influenzinum RC (Roberto Costa), thus confirming absence of
infectious virus in dilution 30d. The viral titer found showed that no viral particles able to cause infection
were present in dilution 30d.

In vitro inhibition of Mycoplasma growth by live nosode

Inhibition halos about 2.0 mm were observed around disks impregnated with live nosode, whereas disks
impregnated with tetracycline exhibited 8.0 mm inhibition halos (Figure 2).

Figure. 2. Inhibition halo in Petri dishes inoculated with Mycoplasma gallisepticum and disk
impregnated with MGR 19610 live nosode 30d (on the left); inhibition halo in Petri dish with Frey
medium inoculated with Mycoplasma gallisepticum and disk impregnated with oxytetracycline
(on the right).

Costa et al [5] concluded that biotherapy was not able to interfere in the growth rate of yeasts when they
developed and assessed the in vitro effects of a new Roberto Costa-type biotherapy against oral candidiasis. In
this study, however, 2.0 mm halos were observed, which were considered non-conclusive.

Protein dosage and polymerase chain reaction (PCR)

Protein dosage by means of Folin-Lowry method showed absence of protein in dilutions 12d and 30d (Fig. 3).

150
Amount of protein (ug/mg)

100

50

0
Mother solution 1st dynamization 1d 12th dynamization 30th dynamization
12d 30d

Figure 3: Protein dosage in mother solution and dilutions 1d, 12d and 30d of MGR 19610 live nosode

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No DNA fragments compatible with M. gallisepticum were found in dilutions 12d, 20d and 30d, whereas
microorganism DNA was found in dilutions 1d, 5d, 10d, 11d and mother solution (MS) (Table 2).

Table 2: Presence of microbial DNA residues in different dilutions of MGR 19610 live nosode.

Dilutions Result
MS +
1d +
5d +
10d +
11d +
12d -
20d -
30d -

Reduction of protein amount with dilution increase was expected, since there is serial decimal dilution from
mother solution onwards, reaching landmark 0.00 μg/mg (absence of measurable protein) from the 12 th
dilution (12d) onwards, where there is neither presence of microbial DNA nor microorganism growth.

These results agree with the findings of several authors, such Arenales, [16] who studied the viability of
homeopathy in veterinary medicine applied to fowl production and concluded that homeopathic medication is
exclusively energetic, since there is no matter in medicines, and Siqueira, [15] who studied cell alterations
induced by a new live-type nosode on lines MDCK e J774.G8, and concluded that readings above 4d were all
similar up to 30d and also similar to water 1d, which confirms that there are not anymore viral
macromolecules such as nucleic acids or proteins in this dilution. Already Hahnemann had stated the
medicines become more powerful when they are diluted and agitated, because this process increases the
medicine efficacy, although from the theoretical point of view molecules are no longer present. [17]

Conclusions

On the grounds of this study results, we conclude that MGR 19610 live nosode may be administered to fowls
in dilution 30d with no risk of causing infection, since there was no in vitro microbial growth from dilution
12d onwards and the following dynamizations eliminate the presence of matter detectable in protein dosage
and PCR assays.

References

[1] Brazilian Homeopathic Pharmacopeia. 2º ed., vol 1. São Paulo (Brazil): Atheneu: 1997. [portuguese]

[2] Costa RA. [Updated homeopathy: Brazilian school] Homeopatia atualizada: Escola brasileira.
Petrópolis(Brazil): Vozes; 1980. [portuguese]

[3] Costa RA. [Live nosodes] Nosódios vivos. Rio de Janeiro: Farmácia Homeopática Átomo; 2002. [portuguese]

[4] Almeida LR. [Research in biotherapy] Pesquisa em bioterápicos. Cult Homeop. 2006;16:12. [portuguese]

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[5] Costa BGB, Siqueira CM, Veiga, VF, Soares RMA, Holandino C. [Development and in vitro assessment of
a new Roberto Costa-type biotherapy for oral candidiasis] Desenvolvimento e avaliação in vitro de um novo
bioterápico do tipo Roberto Costa para candidíase oral. Braz Homeop J. 2009;11(1):15-16. [portuguese]

[6] Costa RA. [Updated homeopathy: Brazilian school] Homeopatia atualizada: Escola brasileira. 3rd ed.
Petrópolis (Brazil): Vozes; 1988. [portuguese]

[7] Nascimento ER, Pereira VLA, Nascimento MGP, Barreto ML. Avian mycoplasmosis update. Braz J
Poultry Sci. 2005;7(1):1-9.

[8] Nascimento ER, Pereira VLA. Mycoplasmosis. In: Fowl diseases. Micoplasmoses. In: Berchieri JR et al, ed.
Doenças das aves. 2ª ed. Campinas(Brazil): Facta; 2009. [portuguese]

[9] [National Agency for Sanitary Surveillance (ANVISA) National Program of Analysis of Residues of
Veterinary Medicines in Foodstuffs Exposed to Consuption – PAMVET] Agência Nacional de Vigilância
Sanitária (ANVISA). Programa Nacional de Análise de Resíduos de Medicamentos Veterinários em Alimentos
Expostos ao Consumo – PAMVET .Brasília (Brazil); 2003. [portuguese]

[10] Benez SM. [Handbook of veterinary homeopathy: clinical and pathological indications, theory and
practice] Manual de homeopatia veterinária: indicações clínicas e patológicas, teoria e prática. São Paulo
(Brazil): Robe; 2002. [portuguese]

[11] Marinho ML. [Therapeutic action of Mycoplasma agalactiae biotherapy in goats with contagious agalactia
of sheep and goats [doctoral dissertation] Federal Rural University of Pernambuco] Ação terapêutica do
bioterápico de Mycoplasma agalactiae em caprinos com agalactia contagiosa dos ovinos e caprinos
[dissertation]. Recife (Brasil): Universidade Federal Rural de Pernambuco; 2008. [portuguese]

[12] Lowry OH, Rosebrough NJ, Lewis Farr A, Randall RJ. Protein measurement with de folin phenol
reagent. J Biol Chem. 1951;193:265-275.

[13] Sambrook J, Fritsch, EF, Maniatis T. Molecular cloning: laboratory manual. 2ª ed. New York (USA): Cold
Spring Harbor Laboratory Press; 1989.

[14] Zeugin JA, Hartley JL. Ethanol precipitation of DNA. Focus. 195;7(4):p.1-2.

[15] Siqueira CM, Feo da Veiga V, Couceiro JN, Lyrio C, Quaresma CH. [Development of a new biotherapy
from infectious influenza virus and verification of its in vivo efficacy] Desenvolvimento de um novo bioterápico
a partir do vírus influenza infeccioso e verificação de sua eficácia in vitro. Cult homeop. 2006;16:56.
[portuguese]

[16] Arenales MC. [Viability of homeopathy in veterinary medicine applied to fowl production] Viabilidade da
homeopatia na medicina veterinária aplicada à produção de aves. Ave World. 2004;2(9):52-53. [portuguese]

[17] Cairo N. [Guide to homeopathic medicine] Guia de medicina homeopática. 21st ed. São Paulo (Brazil):
Livraria Teixeira; 1982. [portuguese]

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Comportamento in vitro do bioterápico tipo nosódio vivo

mycoplasmagallisepticum

RESUMO

Como parte do experimento de uma tese de doutorado, um bioterápico do tipo nosódio vivo utilizando o
microrganismo Mycoplasmagallisepticum estirpe R (ATCC 93-08/19610) foi preparado de acordo com o
paradigma desenvolvido por Costa, observando as normas da Farmacopeia Homeopática Brasileira. O nosódio
vivo foi testado in vitro com a finalidade de avaliar a segurança de sua utilização para imunizar galinhas
domésticas (Gallus gallus) contra a infecção pelo microrganismo M.gallisepticum, e também conhecer seu
comportamento sob condições de laboratório. Não houve crescimento de M. gallisepticum no cultivo em meio
de Frey modificado líquido (caldo) e sólido (placa) das diluições 11d, 12d, 20d e 30d, enquanto que as diluições
1d e 10d mostraram crescimento do microrganismo. Foram observados halos de inibição de cerca de 2,0 mm
em torno de discos de papel impregnados com o nosódio vivo em placas de Petri cultivadas com o
microrganismo, enquanto os discos impregnados com o antimicrobiano convencional oxitetraciclina
apresentaram halos de inibição de 8,0 mm. A dosagem de proteína pelo método de Folin-Lowry identificou a
ausência de proteínas nas diluições 12d e 30d, assim como também não foram encontrados traços de DNA
microbiano na prova de PCR para as diluições 12d, 20d e 30d.

Palavras-chave: Bioterápico; Galinhas; Nosódio vivo; Mycoplasmagallisepticum.

Conducta in vitro de nosodio vivo de mycoplasmagallisepticum

RESUMEN

Una etapa de un proyecto de doctorado, en este estudio fue preparado un nosode vivo del microorganismo
Mycoplasmagallisepticumcepa R (ATCC 93-08/19610) según el modelo de Costa y las normas de la
Farmacopea Homeopática Brasileña. El nosode vivo fue testeado in vitro para determinar su seguridad
cuando utilizado para inmunizar aves domésticas (Gallus gallus) contra la infección por este microorganismo
e investigar su conducta en condiciones de laboratorio. Fue observado que M. gallisepticum no creció en medio
modificado de Frey líquido (caldo) y sólido (placa) con las diluciones 11d, 12d, 20d y 30d. Fueron observados
halos de inhibición de aproximadamente 2,0 mm alrededor de discos de papel impregnados con el nosode vivo
en placas de Petri sembradas con el microorganismo, mientras fueron observados halos de inhibición de 8,0
mm en los discos impregnados con el antibiótico convencional oxitetraciclina. La medición de proteínas
mediante el método de Folin-Lowry evidenció ausencia de proteínas con las diluciones 12d y 30d y ausencia de
vestigios de DNA microbiano por PCR con las diluciones 12d, 20d y 30d.

Palabras-clave: Bioterápicos; Pollos; Nosode vivo; Mycoplasmagallisepticum

Licensed to GIRI
Support: CNPq, FAPERJ
Conflict of interest: authors declare there is no conflict of interest
Received: 20 November 2011; Revised: 12 December 2011; Published: 20 December 2011.
Correspondence author: Môsar Lemos, mosarlemos@vm.uff.br .
How to cite this article: Lemos M, do Nascimento ER, Barreto ML, Pereira VLA, da Silva CC, de Souza JB, Barros SS,
Machado LS, Campos CAM. In vitro behavior of Mycoplasmagallisepticum live-type nosode. Int J High Dilution Res
[online]. 2011 [cited YYYY Month dd]; 10(37): 362-368. Available from:
http://www.feg.unesp.br/~ojs/index.php/ijhdr/article/view/531/552

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