INFLUENCE OF THE ORGANOPHOSPHORUS COMPOUND TRICHLORFON ON THE SUSCEPTIBILITY OF MICE TO RABIESABSTRACT
Influence oftrichlorfon on mice inoculated with rabies virus was studied by observing their susceptibility to the disease and by analyzing the death proportion, incubation period, and the clinicai evolution. Trichlorfon was administered subcutaneously at a dose of 100 mg/kg live weight for 5 consecutive days. The variable between the groups was the beginning ofthe trichlorfon administration in relation to the time of virus inoculation (5 days before, day O, and 5 days after); the mice were males and females, divided into sarne proportion. CVS strain and a street virus isolate were used for mice inoculation. Conceming susceptibility, statistically significant differences were found between the contrais and groups of mice inoculated with the street virus. All groups with trichlorfon treatment were found with higher mortality values than the contrai group. Similarly, with the CVS strain, the only statistically significant results were between the groups that had initiated trichlorfon treatment at day O. Only the groups inoculated with CVS virus presented statistically significant differences in relation to the incubation period (group that initiated trichlorfon treatment at day O), and to the clinicai period (all groups in relation to the group that initiated treatment at day O).
KEY WORDS:
Cholinesterase; mice; organophosphorus compound; rabies; trichlorfon.
RESUMO
A influência da administração do triclorfon em camundongos inoculados com vírus rábico foi estudada sob os aspectos: mortos por raiva, períodos de incubação e curso clínico. Analisaram-se três tratamentos, que consistiram na administração subcutânea do produto (100mg/kg de peso vivo), durante 5 dias consecutivos, variando-se o início do tratamento com triclorfon em relação ao momento da inoculação do vírus (5 dias antes, dia O, e 5 dias após), em grupos de camundongos machos e fêmeas, inoculados com vírus CVS ou com amostra "Rua". Diferenças estatisticamente significantes quanto à proporção de mortos entre tratamentos ocorreram entre os grupos inoculados com vírus Rua, onde os grupos que receberam triclorfon apresentaram resultados estatisticamente significantes em relação ao grupo controle. Entre os grupos inoculados com vírus CVS, a única diferença significante na proporção de mortos por raiva foi observada entre o grupo que iniciou o tratamento no dia O e o grupo controle. Somente entr os grupos inoculados com vírus CVS foram observadas diferenças estatisticamente significantes quanto ao período de incubação (grupo que iniciou tratamento com organofosforado no dia O) e quanto ao período de curso clínico (todos os grupos em relação ao grupo que iniciou o tratamento no dia O).
PALAVRAS-CHAVE:
Colinesterase; camundongos; composto organofosforado; raiva; triclorfon.
INTRODUCTION
It has been suggested that nicotinic acetylcholine receptor (AChR) could act as a co-receptor for rabies virus (TSIANG, 1988). In general, a virus receptor is defined as a sup rficially located cellular structure, in which, after binding of the virus to the cell membrane there occurs a relevant biologic response, such as the virai penetration into cell compartment and its replication; these processes include effects of cellular metabolism or even the generation of an immune response and a productive virus-cell interaction corresponds to the virus multiplication cycle (LENTZ et al.,1983; LENTZ, 1985; MARSH & HELENIUS, 1989; PASTORET, 1990;TIGNOR et al, 1984).
Experiments showing the accumulation of rabies vírus at the neuro-muscular junction on dissected mouse diaphragm; competitive binding of cholinergic antagonists a-bungarotoxin and d-tubocurarine and rabies virus to "in vitro" rat myotubes; characteristics of vírus attachment to membranes of chick embryo myotubes; attachment of rabies vírus to affinitypurified _AChR in a solid-phase binding assay have originated the hypothesis of AChR to be a site of preferential binding for rabies virus, and especially the muscle cells, where a large amount of AChR are found, possibly acting as the primary targets in naturally occurring rabies (LENTZ, 1982; LENTZ et al.,1983; LENTZ, 1985; LENTZ et al., 1986; TSIANG, 1988).
On the other hand, acetylcholine is a chemical transmitter of nervous impulse at the neuro-muscular junctions. Acetylcholine is released by the nerve endings in response to a nervous stimulus, its high concentration permits a rapid diffusion through neuromuscular junctions and attachment to the available AChR. The concentration of free acetylcholine is promptly reduced by the enzyme acetylcholinesterase, which is hydrolyzed to choline and acetic acid, thus preventing the improper stimulation of muscles or effector organs (KHAN,1973; PUGH, 1991, SCIVOLETTO & RAMOS, 1982, TAYLOR, 1987).
In respect to cholinesterases, two types of cholinesterases are distinguished, the "true" or specific acetylcholinesterase, which is found in nervous tissues, erithrocytes, and neuro-muscular junctions; and the other, known as the inespecific or "pseudocholinesterase", found in plasma, intestine, liver, skin and others (KHAN, 1973; PUGH, 1991, SCIVOLETTO & RAMOS, 1982).
The primary biochemical effect associated to the toxicity caused by organophosphorus compounds is the inhibition of cholinesterases (W.H.O.,1986). Differences in concentration of organophosphorus compounds causing toxicity occur due to animal species, age, sex, route of administration and nutritional status (ABDELSALAM, 1987; GAINES, 1969; KHAN, 1973; W.H.O., 1986). Effects of organophosphorus compounds on cholinesterases vary according to chemical composition, sometimes inhibiting indistinctly the cholinesterases, or specially the pseudocholinesterasis or even not affecting the cerebral cholinesterases (KHAN, 1973).
The administration of trichlorfon could interfere to a certain degree in the manifestation of the disease, because the blocking of acetylcholinesterase increases the concentration of acetylcholine in neuromuscular junctions, thus elevating the bindings of acetylcholine to AChR. This fact could make the bindings of rabies virus to the AChR more difficult, since the specific ligant competes with the vírus. However, the vírus could bind indirectly to the components of the cell surface, i.e., through mediation of intermediated molecules (MARSH & HELENIUS, 1989). If acetylcholine could act as a mediator for the bindings of rabies virus to AChR, and due to its high concentration at synapsis levei, the presence of acetylcholine could facilitate the virai bindings. The hypothesis that rabies vírus binds to AChR, so there would exist sites of structural analogy between virus and acetylcholine and the possibility of virus binding to acetylcholinesterase could be considered (WUNNER, 1982). The fusion of rabies virus to cells ispH dependent, it occurs.approximately at pH 6.0 (MARSH & HELENIUS, 1989). In this aspect, the microenvironment at the synapses levei could be altered after administration oftrichlorfon by favoring or not the virai fusion to the target cells.
The administration of trichlorfon temporarily altering the synaptic microenvironment could both represent favorable or unfavorable situation for the virai progression to the CNS.
Taking in consideration the facts that rabies virus progresses through the nerve synapses (MURPHY et al. 1973), and the possible use of AChR as the virai receptors, and as organophosphorus compounds act at synapses level causing accumulation of acetylcholine by inhibiting the enzyme responsible for its metabolization, the present study was designed to assess the influence of trichlorfon on the susceptibility of mice to rabies by measuring the death proportion, incubation periods and the duration of the clinical disease.
MATERIAL AND METHOD
Animals: Male and female albino mice, strain CH-3 Rockfeller, weighing 14-18 gram were used for vírus inoculation and treatment with the organophosphorus trichlorfon and for serum neutralization (SN) test.
Vírus: The CVS-31/2 fixed rabies vírus strain, from the Pan American Zoonosis Center, Argentina, was used. After thawing, the vírus was maintained for three intracerebral passages in mice and showing a titer of 104 79 MICLD 50 /0.03 mL. A caníne field ísolate of rabies vírus M64/90, with two i.e. serial passages in mice and with a titer of 103 5 1 MICLD 50 /0.03 mL was also used.
Diluent: A suspension of sterilized distilled water containing 2% of previously heat-inactivated normal equine serum, 1,000 IU/mL of penicillin and 1.25 mg/ mL of streptomycin constituted the diluent used in the experiment.
Conjugate: The immunofluorescent (IF) conjugate was prepared according to the technique of the Pan American Zoonosís Center (8).
Rabies positive serum: For mice SN test, hyperimmunized equine serum prepared by ínoculation of a commercially available PV (Pasteur vírus) straín vaccine was used.
Trichlorfon**: This was administered subcutaneously to mice a dose of l 00mg/kg oflive weight, diluted in sterilized saline solutíon.
IF test: This test ofbrain specimens was according to the method of GOLDWASSER & KISSLING (1958).
Cholinesterases activity: Determination of plasmatic and cerebral cholinesterases activities was made spectrophotometrically, according to the method of ELLMAN et al. (1961).
Statistical analyses: For the analysis of the influence of triclorfon on the mortality provoked by the vírus, the method of difference between two proportions was used. The ANOVA test was applied to measure the variation existing among the incubation period; period of clinical disease and inhibition índex of plasmatic and cerebral cholinesterases. In case of a significant result, the Tukey test was used (REMINGTON & SHORK, 1970; VIEIRA & HOFFMANN, 1989).
Procedures: The experimental groups were constituted of 288 male and female mice, divided into the sarne proportions and kept separately according to the sexes, and randomly distributed into three major groups: "Street vírus", "CVS vírus", and "CO", respectively with 120, 120, and 48 mice. For better understanding of the treatments applied to different subgroups of mice, the day of vírus inoculation was conventionally registered as day O (zero), days before day O were preceded by the (1978) minus sign, and days after day O, with the (+) plus sign. For notation, days before viral inoculation received a negative number, and days after inoculation, a positive number, in an increasing order.
Street vírus group: A 0.05 mL suspension of street rabies vírus was inoculated into the hind footpads of 120 mice at day O, which were divided into following subgroups:
Street vírus /subgroup -4: Thirty mice were treated with trichlorfon during 5 consecutive days; the first treatment was initiated on day -4, and the 5th and the final one was administered on day O.
Street vírus /subgroup O: Thirty mice were treated with trichlorfon during 5 consecutive days; the first treatment was initiated on day O, and the 5th and the final one was administered on day +4.
Street vírus /subgroup +4: Thirty mice were treated with trichlorfon during 5 consecutive days; the first treatment was initiated on day +4, and the 5th and the final one was administered on day +8.
Street vírus /subgroup Control: Thirty mice were given 0.2 mL of distilled and sterilyzed water, during 5 consecutive days through subcutaneous administration; the first treatment was initiated on day O, and the 5th and the final one, on day +4.
CVS vírus group: A 0.05 mL suspension of CVS strain was inoculated into the hind footp_ads of 120 mice at day O, which were divided into following subgroups:
CVS strain /subgroup -4: Thirty mice were treated with trichlorfon during 5 consecutive days; the first treatment was initiated on day -4, and the 5th and the final one was administered on day O.
CVS strain /subgroup O: Thirty mice were treated with trichlorfon during 5 consecutive days; the first treatment was initiated on day O, and the 5th and the final one was administered on day +4.
CVS strain /subgroup +4: Thirty mice of this group were not treated with trichlorfon, because the CVS virus inoculated killed the animals with a mean incubation period of 5 days. This group was not considered for subsequent analysis.
CVS strain /subgroup Control: Thirty mice were given 0.2 mL of distilled and sterilyzed water, during 5 consecutive days through subcutaneous administration; the first treatment was initiated on day O, and the 5th and the final one, on day +4.
Mice inoculated with street or CVS strain were observed for 45 days, all animals found dead during the experiment were submitted to IF test.
Group CO (Control of Organophosphorus): Mice of this group were not inoculated any virus suspension, they were bcluded to act as the control of trichlorfon intoxication. Forty-eight mice were divided into 8 groups, and treated with varying administration offixed dose of trichlorfon. After treatment, mice were sacrificed at different time intervals. For notation purpose, subscript arabic characters were used, separated with a comma (1968). The first character refers to the number of trichlorfon administration; the second one indicates the time interval in hours after the last administration of trichlorfon and the time when blood and brain samples were collected. Blood samples were collected through cardiac punction of anesthetized mice, and immediately after the sacrifice, brains were collected; and both specimens were submitted to cholinesterases determination.
Subgroup CO_0 2 (Control): Six animals subcutaneously received a single administration ofü.2 mL of sterilized distilled water, and 2 hours after, they were anesthetized and the blood was collected and, immediately after the sacrifice, brains were collected for cholinesterases determination.
Subgroup CO,1 2 : Six animals received subcutaneouslya single administration of trichlorfon, and 2 hours afterwards, they were anesthetized and the blood was collected and, immediately after the sacrifice, brains were collected for cholinesterases determination. Subroup CO,1 24 : Six animals subcutaneously received a single administration of trichlorfon, and 24 hours after, they were anesthetized and collected the blood and, immediately after the sacrifice, brains were collected for cholinesterases determination.
Subgroup CO,2 2 4: Six animals subcutaneously received a daily administration of trichlorfon for 2 consecutive days, and 24 hours after, they were anesthetized and the blood was collected and, immediately after the sacrifice, brains were collected for cholinesterases determination.
Subgroup CO,3 24: Six animals subcutaneously received a daily administration of trichlorfon for 3 consecutive days, and 24 hours after, they were anesthetized and the blood was collected and, immediately after the sacrifice, brains were collected for cholinesterases determination.
Subgroup CO4, 24 : Six animals subcutaneously received a daily administration of trichlorfon for 4 consecutive days, and 24 hours after, they were anesthetized and the blood was collected and, immediately after the sacrifice, brains were collected for cholinesterases determination.
Subgroup CO_524 : Six animais subcutaneously received a daily administration of trichlorfon for 5 consecutive days, and 24 hours after, they were anesthetized and the blood was collected and, immediately after the sacrifice, brains were collected for cholinesterases determination.
Subgroup CO_5 45 : Six animals subcutaneously received a daily administration of trichlorfon for 5 consecutive days, and they were observed during 45 days of the experiment and, brains and blood samples were not collected for cholinesterases determination.
RESULTS
The proportions and percentages of mice injected with trichlorfon and which died dueto inoculation of rabies virus and confirmed by IF test are summarized in Table 1.
Statistically significant differences among treatments were found between the groups treated with trichlorfon and inoculated with street virus and the group street virus/control. With the CVS virus group, statistically significant difference was found between the treatments CVS virus/subgroup O and CVS virus/control.
Proportions and percentages of mice dead after inoculation of a fixed CVS rabies virus strain and a street virus, confirmed through immunofluorescence test, according to different schemes of trichlorfon treatrnent. São Paulo, 1997.
The mean incubation period found for mice inoculated with street vírus, according to treatmentl;, were: 16 days for street virus/subgroup -4; 20 days for street virus/subgroup O; 15 days for street virus/ subgroup +4 and 19 days for street virus/control. For the CVS group, the mean incubation periods were 4days for the subgroups CVS/-4 and CVS/O and 5 days for the subgroup CVS/Control {Table 2).
In respect to the period of incubation, a statistically significant result was found to the group inoculated with CVS vírus by means of ANOVA test (P = 0.0153), and Tukey test indicated a significant difference between the subgroups CVS/O and CVS/Control.
Data corresponding to periods of clinicai evolution ofrabies in mice are illustrated in Table 3.
According to ANOVA test, statistically significant results were found for the CVS group; differences were detected between the comparison of subgroups CVS/-4 and CVS/O, and between the CVS/O and CVS/Control.
The data corresponding to trichlorfon treatments in mice and the plasmatic and cerebral cholinesterases activities are summarized in Table 4.
The plasmatic cholinesterase activities were statistically significant only for the treatments (P<0.005), Tukey test has indicated significance between CO 1,2 and CO 1,24, and between CO 1,2 and CO 2, 24. The cerebral cholinesterase activities were statistically significant (P<0.0001), Tukey test indicated differences among the treatments: CO 0,2 and CO 1,2; CO 1,2 and CO 1,24; CO 1,2 and CO 2,24; CO 1,2 and CO 3,24; CO 1,2 and CO 4,24 and CO 1,2 and CO 5,24.
DISCUSSION
There is evidence that AChR act as receptors for the rabies vírus (LENTZ, 1982; LENTZ et al.,1983; LENTZ, 1985; TIGNOR et al., 1984; TSIANG, 1988). The administration of trichlorfon inhibits the acetylcholinesterase activities, provoking accumulation of acetylcholinesterases at the AchR level, with consequent excessive stimulation (KOELLE, 1970; LENTZ et al., 1983).
Among the groups administered trichlorfon, the proportion of mice dead due to inoculation of street rabies vírus was always greater than that of the controls. Similarly, mice inoculated with CVS strain, the proportion of death found to the trichlorfon-treated groups was never inferior to that of the controls. The significant difference found between the subgroups CVS/O and CVS/Control was at threshold significance. According to the results, the influence of trichlorfon increasing the death proportion was found for the groups inoculated bythe Street vírus, in contrast to the CVS inoculated groups. The proportion of death found for the CVS strain (titer of 104· 79 LD50/ 0.03 mL) was greater than the Street vírus (titer of 103· 5 1 LD50 /0.03mL). The total of deaths found for the CVS groups was 60.22% and for. the Street groups, 30.25% (Table 1). Between the controlgroups, the proportion was 50.00% for the CVS groups, and 6.66% for the Street vírus groups. In an experiment made by BIJELENGA & HEANEY (1990), albino mice inoculated with CVS strain or Street vírus through IP route found lower proportion of deaths for the Street vírus, this fact was attributed to the few number of passages in mice of the Street virus and due to its usually low titer.
In respect to incubation periods, significant difference was found among the CVS groups. The CVS/O subgroup presented a mean incubation period lower to those found for the other CVS subgroups, difference was detected only between the subgroups CVS/O and CVS/Control. lt must be recalled that the CVS/O subgroup has received trichlorfon treatment during the whole period of incubation of the virus, since the period of incubation and the period of trichlorfon treatment had coincided. NILSSON et al.(1985) reported different periods of incubation of street rabies virus in mice, isolated from different animal species, varying from 4 to 19 days, demonstrating the great variability of the incubation periods of isolates, even when inoculated by the intracerebral route and usually longer than the CVS strain. This difference intime between the CVS strain and street virus in reaching the CNS could be in part due to the interaction of rabies virus and cellular receptors (CHARLTON, 1988).
Incubation periods (in days) in mice, of a fixed CVS rabies vírus strain anda field street vírus isolate, according to trichlorfon treatments. São Paulo, 1997.
Periods of clinical evolution (in days) in mice, of a fixed CVS rabies vírus strain and a field street vírus isolate, according to trichlorfon treatments. São Paulo, 1997.
Plasmatic (P) and cerebral (C) cholinesterases activities of mice treated with trichlorfon and variation in absorbance and percentages, according to sex. São Paulo, 1997.
Experimental results revealed that CVS strain can penetrate motor and sensitive neurons without previous replication in myocytes (CHARLTON, 1988; COULON et al.,1989), neurons were found infected after 18 hs post-inoculation and, after 3 days, virus had reach spinal medulla and sensorial ganglia (COULON et al., 1989). Primary cultures of myotubes are numerous in AChR and susceptible to infection of CVS strain and street virus, however, only the street virus produced infective particles, suggesting that CVS strain penetrates directly to peripheric nerves without using the myocytes (CHARLTON, 1988; TSIANG, 1988).
If AChR can act, in the host cells, as the receptors for the rabies virus, more AChR could exist in muscles of more susceptible animais, and theoretically, muscles presenting more receptors would attach a greater number of virus partieles. BAER et al.(1990) analyzed the susceptibility to rabies virus of foxes and skunks and quantified the muscarinic and nicotinic AChR in muscles of both the species; foxes were more sensitive for rabies virus than the skunks and the number of nicotinic AChR was significantly higher than in the slunks. Although the muscarinic AChR were more numerous in foxes, differences were not statistically significant.
Many cell lines without AChR are susceptible to rabies virus, demonstrating that AChR are not essential for the virus infection (REAGAN & WUNNER, 1985). The frequent observation that rabies virus particles accumulate at the synapses level, which contain great concentration of ganglyosides, has strengthened the hypothesis of a ganglyosides role in early stages of infection (TSIANG, 1988). REAGAN & WUNNER (1985), however, stated that rabies virus interaction with several cell lines is independent of the AChR, and the fact of AChR acting as receptors for rabies virus at nerve synapses does not exclude the involvement of other structures in rabies pathogenesis.
The observation of clinicai evolution indicated a statistically significant result among the CVS group. The subgroup CVS/O was found with a mean period of clinical evolution longer than the other CVS subgroups. Through intracerebral inoculation in mice, the evolution of clinicai signs varied from 1-9 days, with most frequent being 2-4 days (NILSSON et al., 1968); shorter periods of 1-3 days were also reported (ATANASIU, 1975).
The analysis of differences of mean plasmatic and cerebral cholinesterases activities revealed that significant results exist between the comparisons made with the CO1,2 subgroup, demonstrating the expected short action of trichlorfon; the maximum time of cholinesterases inhibition is 0.25 hours, and the complete reversion time is 6 hours (MURPHY, 1980). In many animais exposed to organophosphorus compounds, there are temporalily observed higher levels of acetylcholinesterases than the pre-exposure levei (KHAN, 1973). Similar results were found in this experiment, corroborating these previous statements, because both plasmatic and cerebral cholinesterases of CO groups presented higher leveis of cholinesterases than the controls.
It was reported that male rats are more resistant than females to parathion and female rats are more susceptible to trichlorfon treatment (KHAN, 1973). In this experiment, the plasmatic and cerebral cholinesterases leveis, although not being statistically significant, were more elevated in females. It seems that females are more sensitive than male mice, because 2 h after administration of trichlorfon, the values of plasmatic cholinesterase activities were 85% for males and 44.9% for females. Cerebral cholinesterase activities were 54% for males and 20% for females. The experimental results showed greater influence of trichlorfon on the parameter "proportion of death," in the group of mice inoculated with street virus; but future experiments are needed in order to investigate the blocking effect of AChR, sex differerences and using different organophosphorus compounds.
REFERENCES
- ABDELSALAM, E.B. Factors affecting the toxicity of organophosphorus compounds in animais. Vet. Buli., v. 57, n. 6, p. 441-8, 1987.
- ATANASIU, P. Animal inoculation and the negri body. In: BAER, G.M. The natural history of rabies. New York, Academic Press, 1975. v.1, p. 373-400.
- BAER, G.M; SHADDOCK, J.H.; QUIRION, R.; DAM, T.V.; LENTZ, T.L. Rabies susceptibility and acetylcholine receptor. Lancet, n. 17, p. 664-665, 1990.
- BIJELENGA, G. & HEANEY, T. Post-exposure local treatment of mice infected with rabies with two axonal flow inhibitors, colchicine and vinblastine. J Gen. Virol., n. 39, p. 381-385, 1978.
- CENTRO PANAMERICANO DE ZOONOSIS. Prueba de anticuerpos fluorescentes para rabia. Buenos Aires, 1975. 24p. (nota técnica, 8, rev 2).
- CHARLTON, K.M. The pathogenesis of rabies. In: CAMPBELL, J.B. & CHARLTON, K.M. Rabies. Norwell: Kluwer, 1988. p.101-150.
- COULON, P.; DERBIN, C.; KUCERA, P.; LAFAY, F.; PREHAUD, C.;FLAMAND,A. Invasion of peripheral nervous systems of adult mice by the CVS strain of rabies virus and its avirulent derivative AvOl. J Viro/., v. 63, n.8, p. 3550-3554, 1989.
- ELLMAN, G.L.; COURTNEY, D.; ANDRES Jr., V.; FEATHERSTONE, R.M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biachem. Pharmacol., v. 7, p. 88-95, 1961.
- GAINES, T.B. Acute toxicity of pesticides. Toxicol. Appl. Pharmacol., v.14, p. 515-534, 1969.
- GOLDWASSER, R.A. & KISSLING, R.E. Fluorescent antibody staining of street and fixed virus antigens. Proc. Soe. Exp. Biol., n. 98, p. 219-223, 1958.
- KHAN, M.A. Toxicity of systemic insecticides. Toxicological considerations in using organophosphorus insecticides. Vet. Rec., n.92, p.411-419, 1973.
- KOELLE, G.B. Anticolinesterasas. In: GOODMAN, L.S. & GILMAN, A. Bases farmacologicas de la terapeutica. Mexico, D.F.: Nueva Editorial Interamericana, 1970. p. 363-377.
- LENTZ, T.L. Is the acetylcholine a rabies virus receptor? Science, v. 215, n. 4529, p. 182-184, 1982.
- LENTZ, T.L.; BURRAGE, T.G.; SMITH, A.L.; TIGNOR, G.H. The acetylcholine receptor as a cellular receptor for rabies virus. Yale J Biai. Med., v. 56, p. 315-322, 1983.
- LENTZ, T.L. Rabies virus receptor. Trends Neurosci., v. 8, n. 8, p. 360-364, 1985.
- LENTZ, T.L.; BENSON, R.J.J.; KLIMOWICZ, D.; WILSON, P.T.; HAWROT, E. Binding ofrabies virus to purified torpedo acetylcholine receptor. Mal. Brain Res., v. 1, p. 211-219, 1986.
- MARSH, M. & HELENIUS, A. Virus entry into animal cells. Adv. VirusRes., v. 36,p.107-151, 1989.
- MURPHY, F.A.; BAUER, S.P.; HARRISON, A.K.; WINN Jr., · W.C. Comparative pathogenesis of rabies and rabies-like viroses. Virai infection and transit from inoculation site to the central nervous system. Lab. Invest., v. 28, n. 3, p. 361-376, 1973.
- MURPHY, S.D. Pesticides. In: CASSARET & DOLL's. Toxicology: the basic science of poisons. 2.ed. New York: Mac Millan, 1980. p. 357-408.
- NILSSON, M.; SUGAY, W.; PASQUALIN, O.L. Diagnóstico da raiva. Observações sobre tempo de incubação e a duração da doença em camundongos, no período de 1960 a 1966. Arq. Inst. Biai., São Paulo, v. 35, n. 1, p. 1-7, 1968.
- PASTORET, P.P. La réceptivité aux infections d'origine virale. Ann. Méd. Vét., v. 134, p. 291-299, 1990.
- PUGH, D.M. The autonomic nervous system. In: BRANDER, G.L.; PUGH, D.M.; BYWATER, R.J.; JENKINS, W.L. Veterinary applied pharmacology & therapeutics. 5.ed. London: Bailliere Tindall, 1991. p. 97-123.
- REAGAN, K.J. & WUNNER, W.H. Rabies virus interaction with various cell lines is independent of the acetylcholine receptor. Arch. Virai., v. 84, p. 277-282, 1985.
- REMINGTON, R.D. & SHORK, M.A. Statistics with applications to the biological and health sciences. Englewood Clifs: Prentice-Hall, 1970. 418p.
- SCIVOLETTO, R. & RAMOS, L. Drogas colinérgicas e anticolinérgicas. In: CORBETT, C.E. Farmacodinâmica. 6.ed. Rio de Janeiro: Guanabara Koogan, 1982, p. 104-108.
- TAYLOR, P. Agentes anticolinérgicos. In: GOODMAN, L.S. & GILMAN, A. As bases farmacológicas da terapêutica. 7.ed. Rio de Janeiro: Guanabara Koogan, 1987, p. 71-85.
- TIGNOR, G.H.; SMITH, A.L.; SHOPE, R.E. Utilization ofhost proteins as virus receptor. In: NOTKINS, A.L. & OLDSTONE, M.B.A. Concepts in virai pathogenesis. New York: Springer-Verlag, 1984. p. 109-116.
- TSIANG, H. Interactions of rabies virus and host cells. In: CAMPBELL, J.B. & CHARLTON, K.M. Rabies. Norwell: Kluwer, 1988. p. 67-100.
- VIEIRA, S. & HOFFMANN, R. Estatística experimental. São Paulo: Atlas, 1989. 179p.
- WORLD HEALTH ORGANIZATION. Organophosphorus insecticides: a general introduction. Geneva, 1986. (Environmental Health criteria, 63).
- WUNNER, W.H. Is the acetylcholine receptor a rabies-virus receptor? Trends Neurosci., v.5, n.12, p. 413-415, 1982.
