Presence of Actinobacillus pleuropneumoniae , Streptococcus suis , Pasteurella multocida , Bordetella bronchiseptica , Haemophilus parasuis and Mycoplasma hyopneumoniae in upper respiratory tract of swine in farms from Aguascalientes , Mexico

Respiratory diseases are one of the most important health problems in pig herds. The porcine respiratory disease complex (PRDC) is the term used to describe pneumonic diseases caused by multiple infectious agents that provoke weight loss in animals or death. In the PRDC multiple pathogens (bacteria and/or viruses) work in combination to induce this respiratory disease. Within this complex, Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Mycoplasma hyopneumoniae are the main bacterial pathogens involved in great economic losses to the swine industry. The aim of this work was to estimate the presence of A. pleuropneumoniae, S. suis, P. multocida, B. bronchiseptica, H. parasuis and M. hyopneumoniae in the upper respiratory tract of pigs in representative swine farms in Aguascalientes, Mexico, using PCR technique. The study was performed in 14 swine farms. We obtained a total of 212 nasal swabs. Near 20% of samples were positive for A. pleuropneumoniae (located in the 79% of farms); 17% were positive for S. suis (in 86% of farms), of these, 3% were S. suis serovar 2; 30% were positive for H. parasuis (93% of farms); 23% of the samples to P. multocida (in 79% of farms); and 19% to M. hyopneumoniae (in 64% of farms). B. bronchiseptica was not detected in this study. The results obtained show that bacterial pathogens of PRDC were present in the upper respiratory tract of pigs in all farms studied; therefore, these pathogens are widely disseminated in pig farms of Aguascalientes, Mexico.


INTRODUCTION
Respiratory diseases are one of the most important health problems in pig herds.Due to the multifactorial nature of these diseases, they are considered as a porcine respiratory disease complex (PRDC).The PRDC is the term used to describe pneumonic diseases is caused by multiple infectious agents, which produce weight loss in animals or death.The PRDC is a major health problem in the current production of pigs [1,2].Within this complex, the bacteria A. pleuropneumoniae, S. suis, P. multocida, B. bronchiseptica, H. parasuis and M. hyopneumoniae are the main bacterial pathogens involved in causing great economic losses to the swine industry [3], and begin to be regarded as important zoonotic agents with significant increase of infectious in humans [4][5][6].
The objective of this work was to estimate the presence of A. pleuropneumoniae, S. suis, P. multocida, B. bronchiseptica, H. parasuis and M. hyopneumoniae in the upper respiratory tract of asymptomatic pigs in swine farms in Aguascalientes, Mexico, using PCR technique.

Sampling Procedures
For this study were selected 14 farms producing pigs in the State of Aguascalientes, Mexico, for convenience and willingness of producers.The sampling period was from June to October 2011.The total number of pigs sampled was 212 (Table 1).The sample size was calculated according to Alvarez et al. [21], for a study in the State of Yucatan, Mexico, to sought A. pleuropneumoniae and M. hyopneumoniae.The nasal swabs were taken by inserting the swab in the nasal cavity of the pig softly, rotating the swab to easily enter the cavity, and withdrawing it in the same way.The samples were store in sterile tubes.The pigs were randomly selected, ensuring sampling of pigs of different ages and weights, covering the entire farm.Each tube was labeled with the number of the farm, followed by a period and the number of tube following a sequential order.The tubes were store in a cooler until reach the laboratory.In the laboratory, the tubes were store at 4˚C until processed.

DNA Isolation
DNA isolation was performed as described by Sam-brook and Russell [22], with Loera et al. [23] modifications.The swabs were cut in an Eppendorf tube with tweezers and scissors sterilized by flame.Subsequently, 1ml of lysis buffer (15% agarose, 0.03 mM Tris, 0.05 M EDTA, and 0.03 mg lisozime•ml −1 , pH 8) was added directly to the tubes and then they were incubated for 30 minutes at 37˚C.After, 100 μl SDS 10% (0.1 v/v), 100 μl NaCl 5M (0.1 v/v) and 5 μl Proteinase K (0.4 mg•ml −1 ; USB Corporation) was added, and the tubes were mixed and incubated for 1 h at 50 ˚C.Then, 500 μl of the supernatant was taken, in which 200 μl of phenol-chloroform-isoamyl alcohol (24:24:1, Gibco BRL) was added.The mixture was vortexed (Fisher) and centrifuged for 5 min at 10,000 g.The supernatant was recovered and 200 μl ammonium acetate (7.5 M) and 200 μl absolute ethanol (1 v) was added, and mixed for 5 min by inversion and allowed to precipitate overnight at 4˚C.Then, samples were centrifuged at 4˚C for 15 min at 10,000 g.The supernatant was discarded and the pellet was washed several times with 100 μl ethanol (70%).Pellets were dried at 37˚C, resuspended in distilled water and kept at −20˚C until use for the PCR reactions.

PCR Reactions
PCR amplifications were carried out in a DNA thermal cycler (TECHNE T-412).PCR against A. pleuropneumoniae was performed as described by Schaller et al. [24], with modifications for MacInnes et al. [3] and Loera et al. [23].The PCR run conditions were: 95˚C for 1 min followed by 30 cycles of 94˚C for 30 s, 54˚C for 30 s and 72˚C for 1 min with a final elongation step at 72˚C for 5 min.Multiplex PCR for detection of S. suis was developed based on the method of Marois et al. [25].Multiplex PCR permitted the simultaneous detection of the S. suis species (primers 16S-195 [s] and 16S-489 [as2]) and serotypes 2 and 1 2 (primers cps2J-s and cps2J-as).The amplification conditions were: 1 cycle at 95˚C for 1 min, 40 cycles at 94˚C for 30 s, 60˚C for 30 s, 72˚C for 1 min and a final elongation cycle at 72˚C for 10 min.The PCR protocol for detection of H. parasuis was used like previously described Oliveira et al. [26] with modifications for MacInnes et al. [3].The PCR run conditions were: 95˚C for 5 min followed by 30 cycles of 94˚C for 30 s, 59˚C for 30 s and 72˚C for 2 min with a final elongation step at 72˚C for 5 min.PCR against toxigenic strains of P. multocida was developed under the conditions of Kamp et al. [27].The amplification conditions were: 1 cycle at 95˚C for 1 min, 32 cycles at 95˚C for 30 s, 65˚C for 60 s, 72˚C for 2.5 min and a final elongation cycle at 72 ˚C for 20 min.The PCR for B. bronchiseptica was performed using the method Hozbor et al. [28], with modifications by Resgister and DeJong [29].The PCR run conditions were: 1 cycle at 95˚C for 1 min, 35 cycles at 94˚C for 60 s, 53˚C for 30 s, 72˚C for 20 s and a final  elongation cycle at 72˚C for 5 min.Last, PCR for detection of M. hyopneumoniae was developed using the method of Cai et al. [30].The amplification conditions were: 1 cycle at 95˚C for 1 min, 35 cycles at 94˚C for 20 s, 60˚C for 30 s, 72˚C for 40 s and a final elongation cycle at 72˚C for 7 min.All PCR reactions contained 0.25 μl 5 U•μl −1 Taq DNA polymerase (Fermentas), and 2 µl template.All amplified products were observed by elec-trophoresis in 1.5% agarose gel, stain with 1 μg ethidium bromide•ml −1 .Images of the gels were captured using the Chemi Doc (Bio-Rad), image analyzer and the software Quantity One (Bio-Rad, California, USA).The primer sequences are shown in Table 2.

RESULTS AND DISCUSSION
As a representative sample, 14 pig farms distributed in all the area of the State of Aguascalientes, Mexico, were selected for this study.This region, located at central of the country, is semi-arid (overage annual precipitation of 550 mm, 80% in the rainy season in summer), with an average annual temperature of 17˚C -18˚C; however, in spring and summer the overage daily temperature is above 30˚C [31].A total of 212 pigs were sampled, with around 15 pigs per farm (Table 1).Pigs were sampled randomly; they had no apparent signs of illness and were in normal production process on farms.Most pigs sampled were 1 -2 years old; even though, reproductive sows with different ages were also sampled.
Of the 212 nasal swabs, 19.8% were positive to A. pleuropneumoniae and distributed in 78.6% of the farms sampled.Almost 17% of the samples were positive for S. suis, with a distribution in 85.7% of farms; of these, 3.3% were S. suis serovar 2 (the most virulent serovar).In the case of H. parasuis, 29.7% of the samples were positive and distributed in 92.9% of the farms.For P. multocida, 22.6% were positive, with a distribution in the 78.6% of the farms.For M. hyopneumoniae, 19.3% were positive, with a distribution in 64.3% of the farms sampled.Finally, no evidence of B. bronchiseptica in nasal swab samples was found in any of the farms studied (Table 1 and Figure 1).Furthermore, 35.7% of farms showed the presence of five bacterial pathogens of PRDC; the same frequency was observed by the jointly presence of four of them.In 21.4% of farms were found three pathogens; and two pathogens were observed in 7.1% of farms.Farms affected by six PRDC bacterial pathogens were not observed, as well as farms free of these pathogens.Like wise, the bacterial pathogens pairs found most frequently in the same farm were the following: S. suis and H. parasuis (85.7% of farms), and with the same frequency (71.4%) the pairs A. pleuropneumoniae and H. parasuis, H. parasuis and P. multocida, and the pair P. multocida and S. suis.
In Mexico, there are several studies on different pathogens of PRDC, mainly on A. pleuropneumoniae [23,[32][33][34][35][36][37][38][39], but there are few field studies on the bacterial pathogens distribution that make up this complex [21,40], which is the biggest health problem faced by pork producers today.One of these studies, conducted by Álvarez et al. [21], estimate the frequency of A. pleuropneumoniae, M. hyopneumoniae and swine influenza virus (SIV) in the State of Yucatan, region located at the Southeast of the country.They found a frequency of 100% for A. pleuropneumoniae and M. hyopneumoniae.Although in our study we found a lower frequency of both pathogens, probably due to regional differences, it is remarkable the high percentage of farms affected by PRDC pathogens observed in both studies, which clearly show the severe respiratory health problems facing pig production in our country.

CONCLUSION
Bacterial pathogens of porcine respiratory disease complex were present in the upper respiratory tract of pigs in all swine farms sampled, in the State of Aguascalientes, Mexico.These results show the great respiratory health problems present in our swine farms, which is a reflection of the current situation of the Mexican field.

Table 1 .
Presence of swine respiratory pathogens in upper respiratory tract of swine and its distribution per farm.

Table 2 .
Primers sequences used in this study.