Application of PCR technique in diagnosis of four respiratory pathogenic bacteria in pigs at the slaughterhouse

Nong Lam University, Ho Chi Minh City 35 Application of PCR technique in diagnosis of four respiratory pathogenic bacteria in pigs at the slaughterhouse Han M. Ly∗, Trinh T. K. Nguyen, Thiep T. X. Dang, & An T. T. Vo Department of Animal Husbandry and Veterinary Medicine, Nong Lam Univsersity, Ho Chi Minh, Vietnam ARTICLE INFO Research Paper Received: February 20, 2019 Revised: April 26, 2019 Accepted: May 27, 2019 Keywords Multiplex polymerase chain reaction (PCR) Pig lungs Respiratory bacteria Slaughterhouse ∗Corresponding author Ly Mai Han Email: 14112089@st.hcmuaf.edu.vn ABSTRACT The polymerase chain reaction (PCR) for Actinobacillus pleuropneumonia (App), Haemophilus parasuis (Hps), Pasteurella multocida (Pm) and Bordetella bronchiseptica (Bb) were performed in pure colonies isolated from 114 lung specimens with lesions collected from the Vissan slaughterhouse in Ho Chi Minh City from July 2018 to May 2019. The aim of the experiment was to identify the four respiratory pathogenic bacteria in pigs at slaughterhouse by using PCR technique. The criteria for evaluating the results included the proportion of positive samples with multiplex PCR and percentage of samples co-infected with 2, 3, and 4 bacteria. Among a total of 114 injured lung samples, 21% of the samples was positive to at least one of the four bacteria, 3 samples (2.63%) were positive for App, 2 samples (1.75%) were positive for Hps, 7 samples (6.14%) were for Pm, and 12 lungs (10.53%) were positive for Bb. One sample (0.88%) was found co-infected with Pm and Hps. Cited as: Ly, H. M., Nguyen, T. T. K., Dang, T. T. X., Vo, A. T. T. (2019). Application of PCR technique in diagnosis of four respiratory pathogenic bacteria in pigs at the slaughterhouse. The Journal of Agriculture and Development 18(3), 35-40. 1. Introduction Respiratory disease in pigs is one of the lead- ing concerns in the livestock industry. The major direct loss effects on the farmer’s economy due to respiratory illnesses include increased mortal- ity and morbidity rate, reduced weight gain, long finishing time, and high consumption. expenses for treatment (de Jong et al., 2014). Usually, viral respiratory diseases (PRRS, CSFV, PCV-2, etc.) or some important bacteria such as Actinobacillus pleuropneumoniae (App), Bordetella bronchisep- tica (Bb) are the primary factors causing dis- eases. However, the immunodeficiency of infected pigs creates favorable conditions for the aris- ing secondary infections of Haemophillus para- suis (Hps), and Pasteurella multocida (Pm) that normally reside in the upper respiratory tract of the animals. The most important respiratory pathogen is P. multocida (de Jong et al., 2014). The App causes severe acute pleuropneumonia with very high mortality rates of up to 80%. In- fectious rhinitis caused by Bb and Pm is common in commercial pig herds. The Hps causes acute in- fection with characteristic of causing multi-serous inflammation. When these infectious pathogens co-infect, they increase the severity of the disease. While isolation is time-consuming and requires good laboratory skills, diagnosis by PCR method helps to provide accurate results, high reliability www.jad.hcmuaf.edu.vn The Journal of Agriculture and Development 18(3) 36 Nong Lam University, Ho Chi Minh City while saving test time and giving faster results. Thus, the objective of this study was to detect the presence of four respiratory pathogenic bacteria in pigs at the slaughterhouse by using the PCR technique. 2. Materials and Methods The experiment was conducted from July 2018 to May 2019 at the laboratory of Department of Veterinary Biosciences and the Veterinary Hos- pital, Faculty of Animal Science and Veterinary Medicine, Nong Lam University. Four bacteria that have significant impact on respiratory dis- eases in pigs, including App, Hps, Pm and Bb were analyzed from 114 swine lung specimens. 2.1. Sample collection Sample collection was performed at the slaugh- terhouse of Vissan company in Ho Chi Minh City. Injured lungs with lesions, such as congestion, haemorrhage, and inflammation were separated from the carcass and stored in separate zip bags to avoid contamination and transported to the laboratory for culture. 2.2. Isolation method Tryptone Soybean Agar (TSA) (Merck Group, Germany) with 5% bovine serum (Gibco, New Zealand) and Nicotinamide adenine dinucleotide (NAD) (Merck Group, Germany) were used to optimize the growth of four bacteria. Before cul- ture, surface of samples and equipment were dis- infected by using an alcohol swab to clean the surface of the lung until surface was dry. The scissors and forceps were heated using an alcohol- burner and allowed to cool down before use. To obtain an uncontaminated tissue, lung samples were cut deeply in small tissues. Direct smear of the newly cut tissue was performed into a Petri dish containing the culture medium and a ster- ile loop to streak the sample was used. Plates were incubated at 370C for 24 h in bacteriolog- ical incubator (Memmert, Germany). If bacteria growth was seen, the colonies were selected based on colony morphology, catalase reaction (Table 1) and Gram stain (the target bacteria have neg- ative Gram stain). The suitable colonies were transferred into the new TSA medium for pure isolation for the next 24 h. 2.3. Preparation of samples for PCR Bacterial DNA samples were extracted from whole cells by using thermal shock. Pure colonies were placed into an eppendorf containing 50 µL of Tris EDTA buffer solution (TBR, Vietnam) and went through heat cycles (10 min, 1000C; 1 min, - 200C). Cell debris was removed by centrifugation at 12000 rpm in 2.5 min. The supernatant was used directly for PCR process or stored at -200C. The total volume for m-PCR of App, Pm and Hps was 20 µL. The mixture contained 10 µL of Gotaq G2 Green MasterMix, 2 µL of Nulease- Free water (Promega, USA), 1 µL per each primer x 6 primers (AP-IV (Xiao et al., 2006), KMT1 (Townsend et al., 1998), HPS (Oliveira et al., 2001)) (Table 2) and 3 µL of DNA samples. Bac- terial DNA samples were isolated directly from pure colonies by thermal shock. The heat cycle was adapted from Hricˇ´ınova´ (2010) research: (1) the initial phase lasted for 5 min at 950C, then the denaturation was performed at 940C for 30 s. The priming phase lasted for 30 s at 580C, fol- lowed by the extended phase (720C, 45 s) and finally the last 10-min process at 720C. The reaction mixture for s-PCR of Bb was 20 µL including 10 µL of Gotaq G2 Green Master- Mix, 1 µL per each primer (Bb-fla (Hozbor et al., 1999)) (Table 2), 6 µL Nulease-free water and 2 µL DNA extracted from the sample. The initial phase lasted for 5 min at 950C, after which the de- naturation took place at 950C for 30 s. The prim- ing phase lasted for 30 s at 580C, followed by the extended phase (720C, 55 s) and finally the last 10-min process at 720C (Xue et al., 2009). There were 30 cycles performed for each reaction by the peqSTAR thermal cyclers (peqLAB Biotechnolo- gie GmbH, Germany). 2.4. Electrophoresis After completing the PCR reaction, 5 µL of each PCR products used for electrophore- sis. Seven µL of 100 bp DNA ladder (Promega) was used to identify the approximate size of the PCR products. The steps were performed in 1% agarose (Promega) at U = 150 V, I = 144 mA for 20 min (Xue et al., 2009). Actinobacillus pleurop- neumonia ATCC 27090 and Pasteurella multo- cida ATCC 12945 were used as positive controls for these two bacteria. Meanwhile, Haemophilus parasuis and Bordetella bronchiseptica isolated from the field were used as positive control af- The Journal of Agriculture and Development 18(3) www.jad.hcmuaf.edu.vn Nong Lam University, Ho Chi Minh City 37 Table 1. Colony morphology of four bacteria on TSA medium after 24-h incubation Name Colony morphology Catalase reaction Actinobacillus pleuropneumoniae Circular, raised, smooth, cloudy white, 1- 1.5 mm in diameter Negative Haemophillus parasuis Circular, raised, smooth, transparent white, the smallest size in 4 bacteria (< 1 mm) Positive Pasteurella multocida Circular, raised, smooth, opaque white, 3- 3.5 mm in diameter Positive Bordetella bronchiseptica Circular, raised, smooth, greyish white, 1-2 mm in diameter Positive ter being analyzed by PCR and genotyed at Nam Khoa Biotek Company Limited. The PCR products were observed under Biorad UV2000 (Finetech, Taiwan). 3. Results and Discussion There were 24 total objective bacteria strains isolated from 114 injured lungs (21.05%) collected at the slaughterhouse of Vissan Limited Com- pany from July 2018 to May 2019. Three iso- lates of App (2.63%), 2 isolates of Hps (1.75%), 7 isolates of Pm (6.14%) and 12 isolates of Bb (10.53%) were found (Table 3 and Figure 1). Figure 1. Proportion of positive samples diagnosed with PCR. 3.1. Proportion of positive samples diagnosed with PCR The results of this study were different from those of other previous ones in different areas. Bb caused atrophic rhinitis when co-infecting with Pm and resulted in the severity of respiratory in pigs. In this study, Bb had the highest incidence with 10.53% (Figure 1). Zhao et al. (2011) found that 652/3506 lung samples were positive with Bb (18.6%). In North India, 8.2% of nasal swabs were positive with Bb by using PCR technique (Kumar et al., 2014). The gel electrophoresis after amplification of Bb is illustrated in Figure 2. Figure 2. PCR product of Bb - fla gene for detection of Bb after electrophoresis process L: Ladder (1000 bps); Well: 1 - 5: DNA purified from field samples after cultivation; Well 6: positive control (235bps); Well 7: negative control. In this study, Pm infection had the second highest proportion of positive samples diagnosed with PCR method (6.14%); however, this fig- ure was lower than those reported by other re- searchers. In 2017, 296/3212 samples (9.2%) were positive with Pm in China (Liu et al., 2017). In other studies, the presence of Pm was found in 74.9% of lung samples collected from a slaugh- terhouse by using m-PCR technique (Hricˇ´ınova´ et al., 2010). In Vietnam, Le et al. (2012) found that in Bac Giang, the percentage of Bb was 17.14% in the cases of 245 samples that were confirmed www.jad.hcmuaf.edu.vn The Journal of Agriculture and Development 18(3) 38 Nong Lam University, Ho Chi Minh City T a b le 2 . P rim er seq u en ces u sed fo r P C R G en e n a m e P rim er n am e S eq u en ce (5’−→ 3’) S ize (b p ) P referen ce A P -IV A P -IV F A T A C G G T T A A T G G C G G T A A T G G 3 4 6 X iao et al. (2006) A P -IV R A C C T G A G T G C T C A C C A A C G K M T 1 K M T 1 T 7 A T C C G C T A T T T A C C C A G T G G 4 6 0 T ow n sen d et al. (1998) K M T 1 S P 6 G C T G T A A A C G A A C T C G C C A C A P -IV R A C C T G A G T G C T C A C C A A C G H P S H P S -F G T G A T G A G G A A G G G T G G T G T 8 2 1 O liveira et al. (2001) H P S -R G G C T T C G T C A C C C T C T G T B b -fl a B b -fl a -F G C T C C C A A G A G A G A A A G G C T 2 3 5 H ozb or et al. (1999) B b -fl a -R G G T G G C G C C T G C C C T A T C positive with porcine reproductive and respira- tory syndrome virus (PRRSV). In North of Cao Bang and Bac Giang in 2010, it was found that 5% of the pig herd had Pmtext (Le et al., 2012). The gel electrophoresis after amplification of Pm is illustrated in Figure 3. Figure 3. PCR product of KMT1 gene PMT gene af- ter electrophoresis processL: Ladder (1000 bps); Well 1-4: DNA purified from field samples after cultiva- tion; Well 5: positive control (460bps); Well 6: nega- tive control. App is the causative pathogen of pleuropneu- monia in pigs. This bacterium can cause severe lung injuries. The results of this study showed that 2.63% of the samples were positive with this bacterium. This percentage was much lower as compared with those of other studies. In Ben Tre province, the prevalence of App was 24.62% (Thanh et al., 2018) while in Can Tho province, this percentage was 25.9% (Giang et al., 2015) and in Kien Giang, the proportion was 27.69% (Thanh et al., 2017). In some Northen provinces such as Bac Giang, 19.59% of samples positive with PRRSV were also positive with App. Ac- cording to Hricˇ´ınova´ et al. (2010), there was 20.5% of lungs from pigs in slaughterhouse posi- tive to App. The gel electrophoresis after ampli- fication of App is illustrated in Figure 4. Hps is known as the bacteria causing Glasser’s disease and an important agent in the porcine respiratory disease complex. In this study, it was found that only 2/114 lung samples (1.75%) were positive with Hps. Hricˇ´ınova´ et al. (2010) found that 1,83% of lung samples from slaughterhouse were positive with Hps. In Thanh Hoa, Hung Yen and Ha Nam, 20/205 samples (9.7%) includ- ing nasal swab, tracheal fluid, heart and lungs of The Journal of Agriculture and Development 18(3) www.jad.hcmuaf.edu.vn Nong Lam University, Ho Chi Minh City 39 Table 3. Positive samples diagnosed with PCR Total sample Total positive sample App Hps Pm Bb Number of samples 114 23 3 2 7 12 Percentage 100% 21.05% 2.63% 1.75% 6.14% 10.53% Figure 4. PCR product of the gene AP-IV for detec- tion of App after electrophoresis process. L: Ladder (1000 bps); Well 1-2: DNA purified from field samples after cultivation; Well 3: positive control (346 bps); Well 4: negative control. Glasser suspected pigs were found positive with Hps (Truong et al., 2018). In China, Zhao et al. (2011) reported that 26.7% samples were found positive with Hps. The gel electrophoresis after amplification of Hps is illustrated in Figure 5. Figure 5. The PCR product of Hps gene, Hps bacte- ria after electrophoresis process L: Ladder (1000bps); Well 1, 2: DNA purified from field samples after cul- tivation; Well 3: positive control (821 bps); Well 4: negative control. The differences in the percentage of positive samples of the four bacteria in different studies may be associated several factors such as hus- bandry conditions, weather, and disease pressure in various areas. The method of collecting sam- ples may also affect the results as the bacteria are frequently isolated in the upper respiratory tract of pigs, but they would cause diseases when in- vading the lower respiratory tract. Another factor that should be considered is pig sources. In pre- vious studies, samples were collected from clin- ically infected pigs, whereas in this study lungs were taken from pigs in the slaughterhouse with no clinical signs. 3.2. Proportion of samples with co-infection of 2, 3, and 4 bacteria There was only 1 lung with co-infection of Hps and Pm (0.88%). Zhao et al. (2011) found the co-infection of Pm and Bb in all 63 pigs with the atrophy of turbinate bones. So far, the co- infection of those four bacteria has been rarely found in previous studies. 4. Conclusions The prevalence of the investigated pathogens and their co-infection were not high because pigs at the slaughterhouse were relatively healthy and had no obvious clinical signs. 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