Avian cholera: trials to evaluate the efficacy of doxycycline and colistin administered simultaneously in broiler chickens
Published: October 20, 2011
Source : MD Conde*, YD Huberman, HR Terzolo - INTA EEA Balcarce, Buenos Aires, Argentina
Summary
Pasteurella multocida is the etiologic agent of Avian Cholera. This work was designed in order to evaluate the efficiency of the use of Colistin and Doxycycline after an experimental infection with two regional strains of P. multocida, previously isolated from cases of Avian Cholera in Argentina, and also to draw a comparison to the international reference strain X73. For this purpose, a total of 66 broiler chickens of 7 days of age were divided into 6 groups of 11 chickens each. Two groups were injected subcutaneously with one strain; one group was treated with antibiotics in drinking water, while the other group that was injected the same strain remained as untreated control. Mortality was reduced significantly in the tree groups treated with antibiotics, compared to the untreated control groups. It was demonstrated that the combined use of Colistin and Doxycycline had an immediate effect against infections with virulent strains of P. multocida.
Key words: Colistin, Doxycycline, Experimental infection, Pasteurella multocida.
Introduction
Pasteurella multocida is the etiologic agent of Avian Cholera. The presentation of this disease may be acute, subacute, or chronic. The acute or subacute presentation is associated with high mortality in up to 100% of the birds, while chronic cases are associated with local infection in sensorial appendices, infra-orbitary sinuses, peritoneum, liver, lungs, and ovaries (Boyce et al., 2004). As a result of this persistence in chronic cases, birds remain as carriers, resulting in reduced production and decrease in egg fertility. Prophylaxis through vaccination should be considered in areas where Avian Cholera is prevalent, using live or inactivated vaccines (Glisson et al., 2007). However, vaccines do not provide full protection, and it is often necessary to administer antibiotics to prevent septicemia, being careful so as not to generate resistance, especially when antibiotics are administered during long periods of time (Glisson et al., 2007).
Doxycycline is an antibiotic of the tetracycline group. When administered orally, it shows a very rapid and almost complete gastrointestinal absorption, which varies between 93% and 98% of the dose, due to its high solubility in lipids (Goodman & Gilman, 1982), reaching maximum blood concentration between the 2nd and the 4th hour post-administration. This rapid absorption is not affected by simultaneous intake of food. On the other hand, Colistin (or E Polymixin) is a mixture of the Colistin A and Colistin B cyclic polypeptides. When Doxycycline and Colistin are applied simultaneously, both antibiotics are effective against all Gram-negative bacteria, including P. multocida. Previous research, performed in vitro, has shown a sensitivity to Doxycycline in 95% of the P. multocida strains of avian origin studied (Semjen et al., 1998) and also the effectiveness of the administration of Doxycycline in drinking water against an experimental infection with the X73 strain (serotype 1 international reference strain) of P. multocida (Sellyei et al., 2009) has been tested in 5-week-old chicks.
This work was designed to evaluate the efficacy of the Totalbiotic® (Vetanco S. A., Argentina) product, consisting of a combination of Colistin and Doxycycline, against an experimental infection with two regional strains of P. multocida, isolated from cases of Avian Cholera in Argentina and, in comparison with the above mentioned X73 international reference strain.
Materials & Methods
Birds
7-day-old Cobb 500 broiler chickens were used. A sampling of meconium in all birds was performed to ensure that they were free of Salmonella spp.
Birds
7-day-old Cobb 500 broiler chickens were used. A sampling of meconium in all birds was performed to ensure that they were free of Salmonella spp.
Food and water
The birds received water and feed ad libitum. Feed was prepared without the addition of antibiotics or anticoccidians, free of animal and fish meal. Feed was also free of B1, B2, G1 and G2 aflatoxins, Ochratoxin A, T-2 toxin and zearalenone. Bacteriological analyses were performed to ensure that there was no Salmonella spp. in food and drinking water.
The birds received water and feed ad libitum. Feed was prepared without the addition of antibiotics or anticoccidians, free of animal and fish meal. Feed was also free of B1, B2, G1 and G2 aflatoxins, Ochratoxin A, T-2 toxin and zearalenone. Bacteriological analyses were performed to ensure that there was no Salmonella spp. in food and drinking water.
Facilities
The birds were housed in special booths for pathogen discharge. Each booth is made of concrete with glass doors, with positive air pressure to prevent the possibility of cross contamination by air from one booth to the others. The boots have temperature and light control. Water was provided in plastic troughs and feed was provided in linear feeders.
The birds were housed in special booths for pathogen discharge. Each booth is made of concrete with glass doors, with positive air pressure to prevent the possibility of cross contamination by air from one booth to the others. The boots have temperature and light control. Water was provided in plastic troughs and feed was provided in linear feeders.
Strains of Pasteurella multocida
We used two regional strains of P. multocida (1019 and 2088), isolated from cases of Avian Cholera, and an international reference strain (X 73). Prior to these trials, the three strains underwent two passages in 1-day-old chicks through subcutaneous inoculation and subsequent re-isolation of strains in the livers of birds. Then, the re-isolated strains were preserved in vials containing a cryogenic media and frozen by immersion in liquid nitrogen.
We used two regional strains of P. multocida (1019 and 2088), isolated from cases of Avian Cholera, and an international reference strain (X 73). Prior to these trials, the three strains underwent two passages in 1-day-old chicks through subcutaneous inoculation and subsequent re-isolation of strains in the livers of birds. Then, the re-isolated strains were preserved in vials containing a cryogenic media and frozen by immersion in liquid nitrogen.
Innoculum
The vials containing the strains preserved in liquid nitrogen were thawed and the strains that were recovered were planted on Columbia base agar plates, with the addition of 7% of defibrinated bovine blood (ASC). The plates were incubated for 18 hours at 37 º C and from each plate a culture of 10 ml was transferred to brain heart infusion broth. This broth was incubated for 18 hours at 37 º C and was directly used to inoculate the birds with 0.1 ml, subcutaneously. Bacterial enumeration of the broth was performed according to the technique of Miles & Misra (1938).
The vials containing the strains preserved in liquid nitrogen were thawed and the strains that were recovered were planted on Columbia base agar plates, with the addition of 7% of defibrinated bovine blood (ASC). The plates were incubated for 18 hours at 37 º C and from each plate a culture of 10 ml was transferred to brain heart infusion broth. This broth was incubated for 18 hours at 37 º C and was directly used to inoculate the birds with 0.1 ml, subcutaneously. Bacterial enumeration of the broth was performed according to the technique of Miles & Misra (1938).
Administration of antibiotics
The Totalbiotic® commercial product was used: Doxycycline (as Hiclate), 25% and Colistine (as sulfate), 12.5%. The administration of this commercial product took place by dissolving it in drinking water to provide a Doxycycline dose of 10 mg/kg of live weight and a Colistin dose of 5 mg/kg of live weight, which corresponds to a total of 4 g of the product by 10 liters of drinking water. The antibiotics were administered immediately after inoculation.
The Totalbiotic® commercial product was used: Doxycycline (as Hiclate), 25% and Colistine (as sulfate), 12.5%. The administration of this commercial product took place by dissolving it in drinking water to provide a Doxycycline dose of 10 mg/kg of live weight and a Colistin dose of 5 mg/kg of live weight, which corresponds to a total of 4 g of the product by 10 liters of drinking water. The antibiotics were administered immediately after inoculation.
Experimental Design:
The chicks were divided into 6 groups of 11 birds each. For each strain 22 chicks, divided into 2 groups, were challenged: A group of 11 birds received the antibiotic in drinking water, while the other group of 11 birds remained as control group, without administration of the antibiotic. Mortality was recorded during 48 hours post inoculation (table 1), and the difference between the groups was assessed by means of Fisher's test.
The chicks were divided into 6 groups of 11 birds each. For each strain 22 chicks, divided into 2 groups, were challenged: A group of 11 birds received the antibiotic in drinking water, while the other group of 11 birds remained as control group, without administration of the antibiotic. Mortality was recorded during 48 hours post inoculation (table 1), and the difference between the groups was assessed by means of Fisher's test.
Table 1. Mortality caused by Avian Cholera. The birds were injected subcutaneously with 0.1 ml of the innoculum. Mortality was recorded up to 48 hours post inoculation.
|
Strain
|
Dose (CFU)
|
Group
|
Antibiotic*
|
n=
|
Dead
|
Mortality
|
|
1019
|
1.52 x 108
|
1
|
Yes
|
11
|
0
|
0% a
|
|
2
|
No
|
11
|
10
|
90.9% b
|
||
|
2088
|
4.8 x 107
|
3
|
Yes
|
11
|
2
|
18.2% a
|
|
4
|
No
|
11
|
11
|
100% b
|
||
|
X73
|
2.6 x 107
|
5
|
Yes
|
11
|
0
|
0% a
|
|
6
|
No
|
11
|
9
|
81.8% b
|
* Combination of Doxycycline and Colistine (Totalbiotic®, Vetanco S. A., Argentina). Administered at a rate of 4 g per 10 liters of drinking water
a,b Mortality caused by each strain with different lyrics differs significantly (Fisher test, p < 0.05)
a,b Mortality caused by each strain with different lyrics differs significantly (Fisher test, p < 0.05)
Results and Discussion
The mortality rate significantly decreased in the groups treated with antibiotics, compared to birds that did not receive antibiotics in drinking water. In the groups subject to treatment and exposed to strains 1019 and X73, no mortality was recorded, while in the group challenged with strain 2088 2 dead birds were found, from which no P. multocida strains were isolated.
The use of antibiotics against bacterial infections in poultry farming is a very common and important tool. Its was of administration in drinking water is preferable, in order to achieve a rapid and uniform treatment of the infected batch (Vermeulen et al., 2002). In acute cases of Avian Cholera, it is very important to have an antibiotic with immediate effect to prevent septicemia, which can generate a mortality rate of up to 100% of the birds. Previous work has shown the sensitivity of most P. multocida strains to Doxycycline and Colistine. (Jonas et al., 2001; Shivachandra et al., 2004; Sellyei et al., 2009). Furthermore, Semjén et al. (1998) found that Doxycycline lowered the mortality rate in broiler chickens that had been infected (via intramuscular injection) with 2 x 103 CFU/dose of the X 73 strain. In line with this author, this work demonstrated the efficacy of the antibiotic against an infection caused by the same X73 strain, but with a dose (administered subcutaneously) much higher (2.6 x 107 CFU), as well as their effect against experimental infections caused by two pathogenic regional strains of P. multocida.
Conclusions
The use of Totalbiotic® showed immediate action against experimental infections caused by two regional pathogenic strains of P. multocida, achieving a significantly low mortality rate or zero mortality in comparison to untreated birds. Therefore, it can be concluded that the use of Colistin and Doxycycline combined, according to the manufacturer's recommendations, is appropriate to treat cases of Avian Cholera.
Bibliography
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Glisson JR, Hofacre CL, Christensen JP. 2008. Pasteurellosis and Other Respiratory Bacterial Infection: Fowl Cholera, pp. 739-758. En Diseases of poultry 12th ed, Saif YM, Fadly AM, Glisson JR, McDougald LR, Nolan LK, Swayne DE. Blackwell publishing Ames, IA, USA.
Goodman & Gilman. 1982. Las Bases Farmacológicas de la Terapéutica. Ed. Médica Panamericana, México.
Jonas M, Morishita TY, Angrick EJ, Jahja J. 2001. Characterization of nine Pasteurella multocida isolates from avian cholera outbreaks in Indonesia. Avian Dis. 45(1):34-42.
Miles AA & Misra SS. 1938. The estimation of the bactericidal power of the blood. J Hyg. 38:732-749.
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Shivachandra SB, Kumar AA, Biswas A, Ramakrishnan MA, Singh VP, Srivastava SK. 2004. Antibiotic sensitivity patterns among Indian strains of avian Pasteurella multocida. Trop Anim Health Prod 36(8):743-750.
Smejen G, Magyar T, Laczay P. 1998. Therapeutic efficacy of doxycycline against experimental Pasteurella multocida infection in broiler chickens. Acta Vet Hung. 46:85-93.
Vermeulen B, De Backerb P, Remona JP. Drug administration to poultry. 2002. Adv Drug Deliv Rev 54:795-803.
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