The Effect of Furazolidone and Other Drugs on Artificially Induced Escherichia coli Infection in Chickens

ENERGY SOURCE AND PIGEON PERFORMANCE Fisher, H., P. Griminger, G. A. Leveille and R. Shapiro, 1960. Quantitative aspects of lysine deficiency and amino acid imbalance. J. Nutrition, 71: 213-220. Fisher, H., and P. Griminger; 1967. Cholesterollowering effects of certain grains and of oat fractions in the chick. Proc. Soc. Exp. Biol. Med. 126: 108-111. George, J. C , and D. Jyoti, 1955. The lipid content and its reduction in the muscle and liver during

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long and sustained muscular activity. J. Animal Morphol. Physiol. 2: 2-9. George, J. C , and N. V. Vallyathan, 1964. Effect of exercise on free fatty acid levels in the pigeon. J. Appl. Physiol. 19: 619-622. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. Visscher, M. B., 1938. Fat metabolism of the isolated heart. Proc. Soc. Exptl. Bio. Med. 38: 323-325.

THEADOR J. HEBERT AND TIMOTHY S. CHANG The Norwich Pharmacol Company, Norwich, N. Y. 13815 (Received for publication July 3, 1969)

S

INCE the development of the broiler industry following World War II, chronic respiratory disease complex has been a major cause of economic loss through morbidity, mortality, and processing plant condemnations. In 1966 over 32 million pounds of live poultry and 323 million pounds of dressed poultry were condemned because of infectious diseases. In 1967 the losses increased; 33 million pounds of live poultry and 367 million pounds of dressed poultry were condemned and destroyed. Most condemnations were due to leukosis and chronic respiratory disease complex. Total annual losses due to air sac disease in the United States were estimated to be $125 million, or 3 percent of the total value of the poultry industry (Harms, 1968). Although Escherichia coli frequently has been considered to be a secondary invader in the CRD complex, its primary etiologic role has become more apparent. Since most broiler breeders have established mycoplasma-free flocks, an increased number of pure E. coli infections have been reported in the field.

Escherichia coli is classified on the basis of its 0 antigens from the cell itself, K antigens from the capsule or envelope, and H antigens from the flagella. Many serotypes of E. coli have been isolated from diseased poultry (Afnan, 1968; Glantz et al., 1962; Sojka and Carnaghan, 1961). Only a few E. coli serotypes are pathogenic and able to produce disease under artificial conditions. Gross (1956, 1957, 1958), Savov and Paulov (1965) and Hemsley et al. (1967) used Members of 0 groups 1, 2 and 78 experimentally to produce airsacculitis, pericarditis and salpingitis in poultry. Afnan (1968) reported a newly discovered pathogenic serotype of E. coli, 088:k(B)?:H10, on poultry farms in Iran. Gross (1961a) and Sojka (1965) controlled E. coli infection, following inoculation of chickens with serotype 02:K1, by treatment with furaltadone or furazolidone. Craig (1968) considered E. coli as becoming a primary pathogen, or a limited primary invader. He predicted that E. coli alone would continue to cause "air

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The Effect of Furazolidone and Other Drugs on Artificially Induced Escherichia coli Infection in Chickens

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T. J. HEBERT AND T. S. CHANG

EXPERIMENTAL PROCEDURE

Battery experiments were conducted using 2-wk.-old straight-run broiler chicks. Each chick was inoculated with a 24 hr. surface culture of E. coli resuspended in brain-heart infusion broth. One ml. of inoculum was introduced into the posterior thoracic air sac of each chick. Each experiment consisted of infected unmedicated, uninfected unmedicated, and infected medicated groups. Medicated rations were given ad libitum throughout the experiments (13 days). Mortality related to the E. coli infection was recorded during the experimental period. All chickens surviving at the end of medication (13th day) were necropsied and chickens showing E. coli lesions, e.g. airsacculitis, pericarditis, or perihepatitis were considered "condemned." Furazolidone was used in broiler starter feed at 0.011% level in 176 and at 0.022% level in 43 prophylaxis battery experiments. All chicks were challenged with the E. coli (serotype 02a:Kl:H5) inoculum (at 3X10 7 cells/bird) on the third day of experimentation. Medication was given three days before and continuing for ten days after inoculation. The efficacies of furazolidone, oxytetracycline, chlortetracycline, bacitracin methylene disalicylate, zinc bacitracin, erythromycin, procainepenicillin, and penicillin-streptomycin as feed medication, and tylosin as water medication were compared in three prophylactic battery experiments. The drug levels listed in Table 2 were given for 13 days. Each of the 30 chicks in the infected groups were challenged with a similar -E. coli inoculum (at 2X10 7 or 5X10 7 cells/bird) on the third day after the experiment initiation. The experiments were terminated and necropsies were performed at the 13th day. The seven antibiotics and furazolidone

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sac disease" losses. Biddle and Cover (1957) isolated E. coli from 57% of exudates from chickens affected with CRD. Bierer (1962) observed greatly increased death loss in chicks exposed to E. coli and then chilled, as compared to unexposed chicks. Sojka and Carnaghan (1961), Gross (1961a, b), Harry (1964), and Harry and Hemsley (1965a, b) believed that E. coli was a primary cause of many outbreaks of respiratory infection. Gross (1956, 1962) reproduced air sac lesions by injecting E. coli alone or with CRD agent into air sacs of chickens and turkeys. The CRD agent greatly increased the pathogenicity of E. coli when both were given into the same air sac at about the same time. He believed that birds infected with both PPLO and Newcastle disease virus were more susceptible to E. coli bacteremia. Fabricant and Levine (1962) produced experimental CRD by (in descending order of severity): (1) E. coK+PPLO+IBV (infectious bronchitis virus); (2) E. co/i+PPLO; (3) E. coli+IBV; and (4) E. coli alone. Smibert et al. (1960) found mostly E. coli, with PPLO, in natural cases of air sac disease. Bankowski (1961) and Gordon (1961) considered that various combinations of agents would cause respiratory disease. Gale and Baughn (1964) described the standardization of infections with PPLO and with E. coli in chicks, for the testing of treatments. E. coli aggravated changes caused by PPLO and caused disease more readily in PPLO-infected flocks than in PPLOfree flocks, according to Meszaros and Stipkovits (1967). In the work reported here, commonly used medications were tested for efficacy against artificially induced E. coli airsacculitis in chickens.

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E. COLI INFECTION

for the first two weeks of each experiment, and 100 or 200 one-wk.-old chicks were placed in each pen for the respective experiments. In these studies, furazolidone was tested prophylactically against an E. coli inoculum of 1X10 7 cells per ml. There were two control groups in each trial: infected unmedicated chickens, and uninfected unmedicated chickens. All chicks except the uninfected controls were challenged individually by air sac inoculation at 17 days of age. Furazolidone-medicated feed was given at 12 days of age and continued throughout the experiments (8 weeks of age). Necropsy data were collected for all surviving chicks at the termination of the experiment and mortalities were recorded. RESULTS AND DISCUSSION

Chicks inoculated with each of the Ecoli serotypes began to show signs of morbidity approximately 6 hours after challenge. The mortality first occurred between 18 and 24 hours postinoculation and most of the chicks showed airsacculitis at the postmortem examination at this time. However, pericarditis and/or perihepatitis did not occur until 3 to 5 days after inoculation. Bacterial cultures of exudates from these infected chickens revealed E. coli organisms.

TABLE l.—Tlie effect of furazolidone ••In feed in the pirophylaxis of E. coli infection Furazolidone levels

rp . . Treatment

No. of experiments

No. of birds

survivors

0.011%

Furazolidone Infected, Unmed. Uninfected, Unmed.

176 176 176

5,365 5,365 5,365

88 33 100

75- 92 23- 44 100-100

38 73 0

0.022%

Furazolidone Infected, Unmed. Uninfected, Unmed.

43 43 43

1,280 1,280 1,280

97 30 100

85-100 12- 50 100-100

20 75 0

%

99% binomial % survivors condemned limits

Inoculum: 3XlO 7 organisms. Medication: Beginning 3 days before and continuing for 10 days after inoculation. Condemned: Chick showing lesions of either airsacculitis, pericarditis or perihepatitis at the end of medication.

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were compared in one therapeutic battery experiment. Unmedicated ration was given to all chicks for the first three days of the experiment. The feed medication levels listed in Table 3 were initially given to the appropriate groups one half hour after E. coli challenge (2X10 7 cells/bird) and continued for ten days. Chickens were sacrificed at the end of this trial and the incidence of lesions was recorded. For E. coli serotype comparison studies, chickens were inoculated with different serotypes of E. coli in subsequent battery trials. Furazolidone was tested prophylactically at various levels (0.0055%, 0.00825%, 0.011% and 0.022%) against the following serotypes: 02a:Kl:H5, 078:K80:H9, and 01a:K?:H7. The two challenge levels used in the separate trials (3X10 7 or 7X10 7 cells/bird) are listed in Table 4. Experimental procedures followed were similar to those mentioned above for the prophylactic battery trials. Floor pen studies were conducted in concrete floored, windowless poultry houses with forced-air ventilation and perimeter heat at the Norwich Research Center. Fresh wood shavings 2-3 inches deep were used as litter. An equal number of hanging tube type feeders and an automatic waterer were provided in each pen. Infrared brooding lamps were used

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T. J. HEBERT AND T. S. CHANG TABLE 2.—The effect of antibiotics and furazolidone in the prophylaxis of E. coli infection % survivors Treatment (g./ton in feed)

No. birds in each experiment

30 30 30 30 30 30 30 30 30 30 30

Experiment numbers and inoculum (cells/bird) 1 5X10'

2 2X10 7

3 2X10 7

1 5X10'

2 2X10'

3 2X10'

80 63 40 37 33 30 30 13

90 43 27 57 43 53 33 30 33 43 100

100 77 74 47 73 73 44 54

50 47

31 69 50 82 77 62 50 89 82 69 0

13 55 71 46 43 45 60 84

—

3 100

— — — — — — — — —

—

47 100

— 38 0

Medication: Beginning 3 days before and continuing for 10 days after inoculation. •—: Necropsy data were not taken. Condemned: Chicks showing lesions of either airsacculitis, pericarditis or perihepatitis at the end of medication.

The effect of furazolidone on artificially induced E. coli infection in our trials is summarized in Table 1. Furazolidone (0.011%) fed prophylactically allowed 88% survival (99% binomial limits are 75-92) of the infected chicks, while the survival rate in infected unmedicated groups was 33% (99% binomial limits are 23-44). These were results of 176 experiments during the past 7 years. When furazolidone (0.022%) in the feed was used prophylactically, 97% of inoculated chickens survived (99% binomial limits are 85-100) and only 20% of the survivors were condemned (i.e., showed airsacculitis, pericarditis, or perihepatitis at necropsy). Thirty percent of the infected unmedicated controls survived (99% binomial limits are 12-50), and 75% of the survivors were condemned (average from 43 experiments in the past 3 years). These results demonstrated that furazolidone effectively minimized artificially-induced E. coli infection due to the 02a:Kl:H5 serotype. Many antibiotics have been used for prevention or treatment of CRD com-

plex or airsacculitis. In three prophylactic and one therapeutic experiments furazolidone was more efficacious than any of eight antibiotic drugs with which it was compared. Under our experimental conditions, the rate of survival was higher following furazolidone medication than with other medications in each experiment (Tables 2 and 3). A randomized TABLE 3.—The effect of antibiotics and furazolidone in the therapeutic treatment of E. coli infection Treatment (g./ton in feed) Furazolidone (200) CTC (500) OTC (200) ZB (500) Pen-Strep (30+150) Pro-Pen (100) ERY (185) BMD (200) Infected, Unmedicated Control Uninfected, Unmedicated Control

% survivors No. of % birds survivors condemned 30 30 30 30 30 30 30 30

93 70 70 63 60 50 47 47

18 38 24 47 61 40 71 64

30

53

50

30

100

0

Inoculum: 2X10' organisms. Medication: Beginning J hour after inoculation and continuing for 10 days. Condemned: Chicks showing lesions of either airsacculitis, pericarditis or perihepatitis at the end of medication.

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Furazolidone (100) Oxytetracycline (200) Chlortetracycline (100) Bacitracin Methylene Disalicylate (100) Zinc Bacitracin (50) Erythromycin (92.5) Procaine Penicillin (50) Penicillin+Streptomycin (30+150) Tylosin (0.0132% in water) Infected, Unmedicated Control Uninfected, Unmedicated Control

% survivors condemned

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E. COLI INFECTION TABLE 4.—Furazolidone vs. three E. coli serotypes prophytactically

% survivors con
% survivors Treatment

Drug level in feed

(%) 0.005 0.00825 0.011 0.022

— —

1*

2*

3*

Inoculum (cell/bird) 3X10

Furazolidone Furazolidone Furazolidone Furazolidone Infected Control Uninfected Control

3*

2*

1*

No. birds** per serotype

60 60 60 60 60 60

57 72 79 93 27 100

1

3X10 69 68 77 93 62 100

7

7X10 7

3X10 7

3X10 7

7X10 7

—

73 63 57 20 82 0

81 79 61 61 89 0

—

65 85 90 30 100

77 12 38 66 0

blocks analysis on Dunnett's multiple comparison test showed the survival rates with furazolidone medication to be statistically indistinguishable (at the 5% level) from uninfected controls. All the other medications gave survival rates statistically significantly less than control. The same test showed furazolidone and oxytetracycline medications indistinguishable from each other in survival rates, and significantly better (at the 5% level) than infected control and all other medicated groups. The incidence of E. coli lesions generally were lower in furazolidone medicated groups. Results from TABLE 5.—Furazolidone vs. E. cob infection in a prophylactic floor pen study

Treatment

1

Furazolidone 0.011% Infected Control Noninfected Control

100 100 100

92 42 100

11 65 0

2

Furazolidone 0.011% Infected Control Noninfected Control

111

No. of birds

% survivors consurvivors demned

Experiment

90 70 100

8 46 0

.%

Inoculum: 1 X107 organisms. Medication: Beginning 5 days before inoculation and continuing until termination of experiment. Condemned: Chicks showing lesions of either airsacculitis, pericarditis or perihepatitis at the end of medication.

the therapeutic experiment (Table 3) were similar to that of prophylactic experiments. Furazolidone at various levels was effective against other serotypes of pathogenic E. coli (Table 4). Results were similar with E. coli serotypes 078:K80:H9 and 02a: K l : H5. Survival rate rose as the level of furazolidone increased from 0.0055% to 0.022%. However, the incidence of lesions were higher in the experiment with 078:K80:H9 than with 02a:Kl:H5 (Table 4). The effect of furazolidone medication on E. coli serotype 01a:K?:H7 was similar to its effect on 02a:Kl:H5 and 078:K80:H9 (Table 4). Furazolidone medication increased survival and decreased the incidence of disease in surviving chicks. Results from floor pen studies (Table 5) indicated that survival rates in furazolidone-medicated groups were significantly (Chi square) higher than the infected controls (P<0.001). Furazolidonemedicated groups had significantly less condemnation than that of infected control groups (P<0.001). From these results and observations we conclude:

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Expt. 1* Serotype 02a:K1:H5. Expt. 2* Serotype 078:K80:H9. Expt. 3* Serotype 01a:K?:H7. ** 20 birds per group for serotype 01a:K?:H7 (Expt. 3). Medication: Beginning 3 days before and continuing for 10 days after inoculation. Condemned: Chicks showing lesions of either airsacculitis, pericarditis or perihepatitis at the end of medication.

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T. J.

HEBERT AND

1. E. coli serotypes 01a:K?:H7, 02a: K l : H5 and 078: K80: H9 were pathogenic to chickens. 2. Furazolidone was the most efficacious medication against E. coli infection in our experiments. SUMMARY

REFERENCES Afnan, M., 1968. A new pathogenic serotype of Escherichia coli 088:K(B)?:H10. Vet. Rec. 82: 171. Bankowski, R. A., 1961. Respiratory disease complex of chickens in the United States. Brit. Vet. J. 117: 306-315. Biddle, E. S., and M. S. Cover, 1957. The bacterial flora of the respiratory tract of chickens affected with chronic respiratory disease. Am. J. Vet. Res. 18:405^108. Bierer, B. W., 1962. Artificially induced Escherichia coli infection in chilled chicks. Poultry Sci. 41: 1627. Craig, F. R., 1968. E. coli and how you can control it. World's Poultry Sci. J. 24: 141-146. Fabricant, J., and P. P. Levine, 1962. Experimental production of complicated chronic respiratory disease infection ("air sac" disease). Avian Dis. 6: 13-23. Gale, G. O., and C. O. Baughn, 1964. Standardized infections for laboratory evaluation of compounds against a chronic respiratory disease complex in chickens. Poultry Sci. 43:182-186. Glantz, P. J., S. Narotsky and G. Bubash, 1962. Escherichia coli serotypes isolated from salpingitis and chronic respiratory disease of poultry. Avian Dis. 6: 322-328.

CHANG

Gordon, R. F., 1961. Report on avian respiratory disease in Great Britain. Bull. Off. Int. Epiz. 56: 507-529. Gross, W. B., 1956. Escherichia coli as a complicating factor in chronic respiratory disease of chickens and infectious sinusitis of turkeys. Poultry Sci. 35: 765-771. Gross, W. B., 1957. Pathological changes of an Escherichia coli infection in chickens and turkeys. Am. J. Vet. Res. 18: 724-730. Gross, W. B., 1958. Symposium on chronic respiratory diseases of poultry. II. The role of Escherichia coli in the cause of chronic respiratory disease and certain other respiratory diseases. Am. J. Vet. Res. 19: 448-452. Gross, W. B., 1961a. The development of "air sac disease." Avian Dis. 5:431-439. Gross, W. B., 1961b. The effect of chlortetracycline, erythromycin and nitrofurans as treatments for experimental "air sac disease." Poultry Sci. 40: 833-841. Gross, W. B., 1962. Blood cultures, blood counts and temperature records in an experimentally produced "air sac disease" and uncomplicated Escherichia coli infection of chickens. Poultry Sci. 41:691-700. Harms, R. H., 1968. PPLO control, Marek's, other items covered at poultry institute. Feedstuffs, 40(31): 48, 50. Harry, E. G., 1964. A study of 119 outbreaks of colisepticaemia in broiler flocks. Vet. Rec. 76: 443449. Harry, E. G., andL. A. Hemsley, 1965a. The association between the presence of septicaemia strains of Escherichia coli in the respiratory and intestinal tracts of chickens and the occurrence of coli septicaemia. Vet. Rec. 77: 35-40. Harry, E. G., and L. A. Hemsley, 1965b. The relationship between environmental contamination with septicaemia strains of Escherichia coli and their incidence in chickens. Vet. Rec. 77: 241-245. Hemsley, R. V., D. A. Barnum and D. G. Ingram, 1967. Biochemical and serological studies of avian strains of Escherichia coli. Avian Dis. 11: 90-97. Meszaros, J., and L. Stipkovits, 1967. I. Experimental Escherichia coli bacteraemia in chicks. II. Spread of Escherichia coli within flocks infected with Mycoplasma gallisepticum. Magy. Allatorv. Lap. 22: 14-23 (in Hungarian). (Vet. Bui. 38: 69, 1968.) Savov, D., and N. Paulov, 1965. Experimental E. coli infections in chicks. Vet. Med. Nauki. Sof. 2: 695-700 (in Bulgarian). (Vet. Bui. 36: 276, 1966.)

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As broiler growing has increased in the last 25 years, chronic respiratory disease has caused major economic loss through morbidity, mortality, and processing plant condemnations. Escherichia coli usually is isolated from diseased birds. Although it has been considered a secondary invader, its primary role has become increasingly apparent in the field. Certain serotypes of E. coli are prevalent causes of disease in poultry. Furazolidone is more efficacious in controlling artificially induced E. coli infection than eight other medicaments with which it was compared.

T. S.

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E. COLI INFECTION Smibert, R. M., J. E. Faber and H. M. Devolt, 1960. Studies on "air sac" infection in poultry. 3. Bacterial flora of the respiratory system of poultry associated with avian PPLO (Pleuropneumonia-like organisms) in natural cases of aerosacculitis. Poultry Sci. 39: 417-426. Sojka, W. J., 1965. Escherichia coli in domestic

animals and poultry. Rev. Ser. No. 7. Commonwealth Bureau of Animal Health, Weybridge. C. A. B. Sojka, W. J., and R. B. A. Carnaghan, 1961. Escherichia coli infection in poultry. Res. Vet. Sci: 2: 340-352.

Evidence that Taurine May Be One of the Elusive Unidentified Factors W. J. MONSON Borden Inc., Chemical Division, Nutritional Research Laboratory, Elgin, Illinois 60120

' I VHE search for one or more unidenti•*• fied factors in certain feed ingredients has been long and arduous. Many research reports have been written on the benefits of these ingredients in improving growth, egg production, hatchability, and feed efficiency of poultry. It was not a problem to find references to the various phases of this research and no attempt was made to document them in this paper. It is generally accepted by those who believe unidentified factors exist that fish meal and fish solubles are sources of these factors. It is the purpose of this report to show evidence that the compound taurine is at least one of these factors, and for taurine to express its activity, another factor present in a commercial fermentation product, Fermacto 500, is also required. The unidentified factor activity of Fermacto 500 is referred to as the fermentation factor in this report. Martin and Patrick (1961) reported that the addition of 0.1% taurine to a simplified diet utilizing 30% Drackett protein increased the weight of chicks slightly at 7 and 14 days of age but resulted in a slight growth depression at 21 days of age. A subsequent paper by Martin and Patrick (1966) indicated increased weight, feed efficiency, and protein efficiency ratio at 14 days of age as a result of

adding 0.1% taurine to a similar diet. These authors suggested the response was obtained because of a relatively low level of methionine. They also reported little growth response to taurine when using a casein-purified diet with optimal supplementation and less growth promotion from taurine when the soybean protein purified diet was fortified with methionine. Roe and Weston (1965) reported the concentration of taurine in the edible portions of various foods. They found the highest taurine values in sea foods from invertebrate phyla, but the other animal protein foods also contained significant quantities of the amino-acid: It was indicated that foods of vegetable origin have extremely low concentrations or no taurine. The object of these experiments was to determine whether taurine could be a factor needed by chicks for maximum growth rate and further to determine whether this was associated with the unknown factor activity of fish meal, EXPERIMENTAL

There have been fourteen chick battery experiments conducted on this problem at our laboratory from April of 1966 through 1968. A few of these have been selected that are representative of our re-

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(Received for publication July 3, 1969)