Pasteurella Multocida: effect of in vivo passages on pathogenicity in chicks

Introduction

Pasteurella multocida it is the ethiological agent of Avian Cholera. Also, P. multocida may be both a primary pathogen, and a secondary agent associated with other diseases. The course of this condition may be acute, sub-acute, or chronic. The acute or sub-acute form is associated with septicemia and high mortality, while chronic cases are associated with local infection in sensorial appendices, paranasal sinuses, peritoneum, liver, lungs, and ovaries (Boyce et al., 2004). In chronic cases of Avian Cholera, birds usually remain carriers and suffer reduction in egg production, while in breeding birds the disease also causes decrease in fertility. The pathogenicity depends on the strain isolated, the host and his health state (Glisson et al., 2008). Known virulence factors include: the capsule, the LPSs, toxins, the iron acquisition system, adhesins and other membrane proteins. However, the participation of each of these factors has not been entirely clarified (Boyce et al., 2010).

Pasteur (1880) was the first to describe the decline of pathogenicity of P. multocida after passages in artificial culture media and their subsequent recovery after in vivo passages. Therefore, it is not possible to reproduce Avian Cholera without establishing before the adequate inoculation dose. Although some highly pathogenic strains have been described (Chung et al., 2001; Harper et al., 2007), different descriptions on the pathogenicity of local isolates of P. multocida exist.

The objective of this work is to get to know the pathogenicity of local isolates of strains of P. multocida to predict their ability to cause mortality and establish a standard model of experimental reproduction of Avian Cholera which is adequate for evaluation of preventive or curative treatment.

Materials & Methods

Birds
One-day-old male chicks of layer hens of the Hisex brown line.

Strains of Pasteurella multocida
We used 10 regional strains of p. Multocida from the strain pool of the Bacteriology Laboratory of INTA EEA Balcarce (Tabla 1).

Table 1. Strains of Pasteurella multocida

*Group*	*Strain*	*Province*	*Organ*	*Year*
1	1019	Entre Ríos	Sensorial appendix	1999
2	697	Entre Ríos	Ovary	2001
3	827	Buenos Aires	Lung	2001
4	1269	Buenos Aires	Lung	2002
5	1370	Entre Ríos	Peritoneum	2003
6	1734	Entre Ríos	Ovary	2006
7	1814	Entre Ríos	Liver	2007
8	2044	Entre Ríos	Ovary	2009
9	2088	Entre Ríos	Liver	2009
10	2117	Buenos Aires	Liver	2009

Innoculum

The strains were extracted from vials frozen in liquid nitrogen and sown on Columbia Base Agar with 7% bovine blood (ASC). The strains were incubated for 18 hours at 37 º C and from each plate a culture was transferred to 10 ml of brain heart infusion broth. This broth was incubated for 18 hours at 37 º C and was directly used to inoculate the birds.

Experimental Design

Test 1: Of each of the strains, 0.1 ml of inoculum, developed in brain heart infusion broth, were injected subcutaneously in 5 two-day-old chicks . Mortality was recorded up to 24 hours post-inoculation. The livers of chicks that died or were slaughtered by cervical dislocation were extracted (at a rate of 2 chicks per strain) (AVMA, 2007). Two livers of each batch were manually macerated in plastic bags on PBS (pH 7.2) and planted in ASC and Base Agar for re-isolation of P. multocida, as well as in MacConkey Agar, for control of possible pollutant enterobacteria.

Test 2: Of each of the strains, 0.1 ml of the liver macerate obtained in Test 1 was injected subcutaneously into 5 3-day-old chicks. Mortality was recorded up to 24 hours post-inoculation; all the birds that survived were slaughtered. All the livers of chicks that died or were slaughtered in each batch were extracted. The livers were macerated and planted in a form similar to that described in Test 1. Grouping together the livers from each batch of poultry, counts on each macerate were performed, using the Miles and Misra technique (1938), on ASC plates.

Test 3: 5 Strains that had caused death or septicemia in previous trials were selected. These 5 strains were injected subcutaneously in 10 4-day-old chicks, using 0.1 ml of the liver macerate obtained in Test 2. Mortality was recorded up to 24 hours post-inoculation and all the birds that survived were slaughtered. The livers of the chicks that died or were slaughtered were extracted. The livers were planted in a manner similar to that described for Test 1.

Results

In Test 1, using the brain heart infusion broth inoculum, made with the strains extracted from liquid nitrogen, mortality was observed in groups 1 and 9 only (in 1 bird out of 5 and in 2 birds out of 5, respectively (Table 2).

In Test 2, carried out through the use of liver macerate obtained in test 1 as inoculum, mortality was observed in the same two groups (1 and 9). On the other hand, P. multocida was isolated in pure culture of liver macerates from groups 8 and 10, although this bacterium caused no mortality. Also, 2 birds were found dead for reasons not related to the test in Group 4; but P. multocida could not be isolated from the macerate.

In Test 3, only 5 strains selected by their apparent pathogenicity were used: 3 strains that caused mortality (Groups 1, 4 and 9) and 2 strains that caused liver infection, although without fatality (Groups 8 and 10). The trial was performed through subcutaneous injection of 0.1 ml of the liver macerate obtained in Test 2, containing approximately 10⁷ colony forming units of P. multocida per bird, in pure culture. Mortality recorded in Groups 1 and 9 was 100% while in the other 3 inoculated groups only 1 dead chick was found in group 10. P. multocida was isolated from the 5 liver pools.

Table 2. Innoculum and mortality caused by Pasteurella multocida

		*Test 1*	*Test 2*	*Test 3*
Group	Strain	Innoculum^A	Dead	Macerated^B	Dead	Macerated^C	Dead
1	1019	+	1/05	+	4/5	+	10/10
2	697	+	0/5	-	0/5	Not performed
3	827	+	0/5	-	0/5	Not performed
4	1269	+	0/5	-	2/5	+	0/10
5	1370	+	0/5	-	0/5	Not performed
6	1734	+	0/5	-	0/5	Not performed
7	1814	+	0/5	-	0/5	Not performed
8	2044	+	0/5	+	0/5	+	0/10
9	2088	+	2/5	+	5/5	+	10/10
10	2117	+	0/5	+	0/5	+	1/10

^{A - Brain heart infusion broth innoculum made from the strains extracted out of liquid nitrogen
B - Liver macerate obtained in Test 1 in PBS
C - Liver macerate obtained in Test 2 in PBS}

Discussion

All the regional strains used were isolated from clinical cases of Avian Cholera and kept frozen in liquid nitrogen for several years. According to the early work of Pasteur (1980), the results obtained in this study showed an increase of the pathogenicity of some strains of P. multocida studied; thus it was demonstrated that, after two in vivo passages in Group 1 (strain 1019) mortality increased from 20% to 100% and in Group 9 (strain 2088), from 40% to 100%. However, some of the strains that were studied showed ability for liver invasion, although they were able to cause little or no mortality only. These recent strains were generally associated to chronic cases of Avian Cholera, generating losses in form of low production and fertility but without causing mortality in farms. Nevertheless, the possibility exists that if a greater number of in vivo passages had been done, the pathogenicity of these strains could have increased.

Conclusions

The pathogenicity of native strains of P. multocida and changes in virulence after two in vivo passages were studied. These results provide relevant information to establish a standard model of experimental reproduction of Avian Cholera for the purpose of evaluation of regional strains.

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