Mycoplasma hyopneumoniae can be an important pathogen in porcine respiratory disease complex¹ as well as the primary pathogen of enzootic pneumonia, a chronic respiratory disease in growing pigs resulting from combined infections of M hyopneumoniae and one or more secondary bacterial pathogens.² Enzootic pneumonia is characterized by a persistent nonproductive cough with a reduced growth rate, a poor feed conversion ratio, high morbidity, and low mortality.^3,4 The economic impact of M hyopneumoniae infections in swine farms worldwide can be considered significant.

Several strategies may be implemented to successfully prevent and control M hyopneumoniae including optimized management practices and vaccination.⁵ While all-in/all-out production and multisite operations are great management tools, vaccination remains an important and cost-effective method for reducing the impact of M hyopneumoniae infection. The M hyopneumoniae-free status of herds is difficult to maintain especially in pig-dense areas, since the airborne spread of this pathogen may occur over several kilometers.⁶

In Korea, approximately 70% of total piglets farrowed in 2018 were vaccinated with M hyopneumoniae (http://www.kahpha.or.kr). Therefore, vaccination is one of the tools used to control M hyopneumoniae. The objective of this study was to evaluate the efficacy of a new single-dose M hyopneumoniae whole-cell bacterin (Hyogen, CEVA Santé Animale) based on strain BA 2940–99, oil adjuvanted with paraffin and Escherichia coli J5 LPS with thiomersal as excipient under field conditions in accordance with the registration guidelines of the Republic of Korea’s Animal, Plant and Fisheries Quarantine and Inspection Agency (http://www.qia.go.kr).

Materials and methods

The protocol for this field study was approved by the Seoul National University Institutional Animal Care and Use Committee (approval number SNU-180621-13).

Farm histories

The clinical field trial was conducted on 3 Korean swine farms (denoted as Farms A, B, and C) between August 2018 and February 2019. Status of porcine reproductive and respiratory syndrome (PRRS) was stable with no active PRRS virus circulation (high-parity sows were the only seropositive animals in the herd). Porcine circovirus type 2 (PCV2) was circulating in the postweaning and growing period without overt clinical signs of porcine circovirus-associated disease on the 3 farms.

Farm A was a conventional 400-sow farrow-to-finish swine farm where the owner complained about a dry recurrent cough beginning at 40 days of age accompanied by growth retardation. Real-time polymerase chain reaction (PCR) testing⁷ of pneumonic and atelectatic lung samples from pigs at 49 days of age was conducted for M hyopneumoniae at the Veterinary Diagnostic Center, College of Veterinary Medicine, Seoul National University in May 2018. The testing returned positive results for 5 of the 7 lung samples submitted for M hyopneumoniae. The combined occurrence of clinical signs, detection of M hyopneumoniae by PCR, and histopathological lesions (peribronchiolar and perivascular lymphoid tissue hyperplasia) were indicative of an ongoing infection with M hyopneumoniae.

Farm B consisted of a conventional 150-sow farrow-to-finish swine farm managed in a 2-week batch system and included a history of enzootic pneumonia. Infection with M hyopneumoniae was evident by severe dry coughing, histopathological peribronchiolar lymphoid tissue hyperplasia, and detection of M hyopneumoniae in lung samples by real-time PCR⁷ in all three of the 38-day-old pigs tested.

Farm C, a conventional 450-sow farrow-to-finish swine farm, was suggested to our clinical study team by its practitioner to participate in this field trial on M hyopneumoniae vaccine efficacy. A pilot survey was implemented to assess the circulation of M hyopneumoniae within the herd, as the producers had complained of severe dry coughing and retardation of growth between 10 and 50 days of age. Lung samples from 74-day-old pigs were submitted to the Veterinary Diagnostic Center, College of Veterinary Medicine, Seoul National University in June 2018. Three of the 5 lung sample submissions were positive for M hyopneumoniae using real-time PCR testing.⁷ The histological lesions were characterized by peribronchiolar lymphoid tissues hyperplasia and bronchopneumonia. Pasteurella multocida was isolated in 4 of the 5 lung samples. These results were indicative of enzootic pneumoniae by M hyopneumoniae with secondary P multocida infection.

Study design

The experimental design of the field study strictly adhered to the registration guidelines set by the Republic of Korea’s Animal, Plant and Fisheries Quarantine and Inspection Agency. Guidelines require that 20 piglets (10 male and 10 female) be selected and assigned to each group of vaccinated and unvaccinated animals. To minimize sow variation, four to six 7-day-old piglets were randomly selected from each sow and assigned to either the vaccinated or unvaccinated group using the random number generator function in Excel (Microsoft Corporation). The pigs in the vaccinated groups were injected intramuscularly in the right side of the neck with 2 mL of the M hyopneumoniae bacterin (Hyogen, CEVA Santé Animale, Lot No.1405582B) at 21 days of age, while an equal volume of phosphate buffered saline (0.01M, pH 7.4) was injected in the same anatomical location for pigs of the unvaccinated groups. At 24 days of age, all vaccinated and unvaccinated pigs were transferred to the nursery facility and kept in co-mingled groups until the end of the trial. In the nursery, pigs were then randomly distributed into 4 total pens to include 10 pigs/pen, all within one room. A similar proportion of each treatment was included in each pen. All pens were identical in design and equipment which included free access to a feed and water trough in accordance with standard farm procedures. The 3 farms did not use feed or water medication effective against M hyopneumoniae. Antibiotics (ie, penicillin) were given to vaccinated and unvaccinated pigs to help control respiratory diseases during the course of the study. Blood and nasal swabs were collected at study days 0 (21 days of age), 21 (42 days of age), 49 (70 days of age), 77 (98 days of age), and 105 (126 days of age).

Mortalities

Pigs that died were subjected to gross pathological examination within 24 hours at a local veterinary practitioner’s clinic. All major organs such as brain, lung, subinguinal lymph node, small and large intestine, liver, kidney, and tonsils were collected from each pig. In the case of lung lesions, samples were collected from the edge of these lesions. Polymerase chain reaction assays were used to detect specific nucleic acids for PCV2, PRRS virus, swine influenza virus, and M hyopneumoniae.^8-11 All other bacterial isolation and identifications were carried out by using routine methods.

Clinical observations

Pig physical condition was monitored daily, and pigs were scored weekly for clinical respiratory disease from study days 0 to 105. Scores ranged from 0 to 6: 0 = normal; 1 = mild dyspnea, tachypnea, or both when stressed; 2 = mild dyspnea, tachypnea, or both when at rest; 3 = moderate dyspnea, tachypnea, or both when stressed; 4 = moderate dyspnea, tachypnea, or both when at rest; 5 = severe dyspnea, tachypnea, or both when stressed; 6 = severe dyspnea, tachypnea, or both when at rest. Observers were blinded to vaccination status.

Growth performance

Pigs were weighed at study days 0 (21 days of age), 49 (70 days of age), 91 (112 days of age), and 154 (175 days of age). Average daily gain (ADG) was determined for study days 0 to 49, study days 50 to 91, and study days 92 to 154 (Table 1). The ADG during these various stages was calculated as the difference between the starting and final weight divided by the duration of the stage. Data for dead or removed pigs were included in the calculation.

Quantification of M hyopneumoniae DNA in nasal swabs

Sterile polyester swabs (Fisher Scientific Inc) were used to swab the nasal mucosa of both nostrils, reaching deeply into the turbinates. Swabs were stored in 5 mL plastic tubes (Fisher Scientific Inc) containing 1 mL of sterile saline solution. A commercial kit (QIAamp DNA Mini Kit, QIAGEN) was used to extract DNA from nasal swabs to quantify the M hyopneumoniae genomic DNA copy numbers by real-time PCR as previously described.⁷ To construct a standard curve, real-time PCR was performed in quadruplicate in 10-fold serial dilution of chromosomal DNA from M hyopneumoniae strain SNU98703, with concentrations ranging from 10 ng/µL to 1 fg/µL. One femtogram of chromosomal DNA from M hyopneumoniae is considered to be approximately one genome equivalent.¹² A negative control was included in each run using double distilled water as the template.

Enzyme-linked immunosorbent assay

Blood samples were collected from each pig by jugular venipuncture. Serum samples were tested for M hyopneumoniae antibodies using a commercial enzyme-linked immunosorbent assay (ELISA; IDEXX Laboratories Inc). Serum samples were considered positive for M hyopneumoniae antibodies if the sample-to-positive (S:P) ratio was ≥ 0.4 in accordance with the manufacturer’s instructions.

Enzyme-linked immunospot assay

Blood samples were collected from each pig by jugular venipuncture. The enzyme-linked immunospot (ELISpot) assay was conducted to measure the number of M hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SC) in peripheral blood mononuclear cells (PBMC).¹³ Mycoplasma hyopneumoniae antigens were prepared as previously described.¹⁴ The IFN-γ positive spots on the membranes (MABTECH) were imaged, analyzed, and counted using an automated ELISPOT Reader (AID ELISPOT Reader, AID GmbH). The results were expressed as the number of IFN-γ-SC per million PBMC. The ELISpot assay was completed in duplicate.

Pathological evaluation

Lung samples were collected in pigs from each group at study day 147 (168 days of age). Lung pathology evaluation was done by two pathologists (authors Oh and Chae) at the Seoul National University (Seoul, Republic of Korea). Macroscopic lesion scores were estimated, and a score was given to reflect the amount of pneumonia in each lobe. For the entire lung, up to 100 points were assigned as follows: 10 points each to the right cranial lobe, right middle lobe, left cranial lobe, and left middle lobe; 27.5 points each to the right caudal lobe and left caudal lobe; and 5 points to the accessory lobe.¹⁵ Eight pieces of lung tissues (two pieces from the right cranial lobe, two from the right middle lobe, one from the ventromedial part of the right caudal lobe, one from the dorsomedial part of the right caudal lobe, one from the midlateral part of the right caudal lobe, and one from the accessory lobe) were collected from each pig. Three tissue sections of the eight lung pieces were examined blindly by two veterinary pathologists (Oh and Chae). Lung sections were scored for presence and severity of type 2 pneumocyte hypertrophy and hyperplasia, alveolar septal infiltration with inflammatory cells, peribronchial lymphoid hyperplasia, amount of alveolar exudate, and amount of inflammation in the lamina propria of bronchi and bronchioles ranging from 0 to 6: 0 = normal; 1 = mild multifocal; 2 = mild diffuse; 3 = moderate multifocal; 4 = moderate diffuse; 5 = severe multifocal; 6 = severe diffuse.¹⁶

Statistical analysis

Prior to statistical analysis, real-time PCR data were transformed to log₁₀ values to reduce variance and positive skewness. The normality of the distribution of the examined variables was evaluated by the Shapiro-Wilk test. Continuous data (ADG, real-time PCR, ELISA, and ELISpot) were analyzed with a Student t test to determine the significance of group differences at each time point. Discrete data (clinical signs and pathology lesions) were analyzed by Mann-Whitney test to determine the significance of group differences at each time point. A P value < .05 was considered significant.

Results

Mortality

One vaccinated pig from farm A died of bronchopneumonia resulting from a combination of PCV2 that was detected with PCR and Glasserella parasuis that was isolated from the lung at study day 51 (72 days of age). Three unvaccinated pigs from farm A died of pleuropneumonia caused by a combination of Actinobacillus pleuropneumoniae and other bacteria. Actinobacillus pleuropneumoniae and P multocida were isolated from lung tissue at study days 74 (95 days of age) and 77 (98 days of age), and A pleuropneumoniae and Streptococcus suis were isolated from lung tissue at study day 93 (114 days of age). Farm C had 1 vaccinated pig die of salmonellosis at study day 42 (63 days of age) and 2 unvaccinated pigs died of bronchopneumonia caused by a combination of PCV2 that was detected with PCR and P multocida that was isolated from lung tissue at study day 72 (93 days of age) and 92 (113 days of age), respectively. But PCV2-associated lesions were not observed in lymph nodes from these 2 pigs.

Clinical signs

Vaccinated pigs from farm A had significantly lower (P = .004) clinical respiratory scores when compared with unvaccinated pigs at study days 21 to 56. Farm B vaccinates also had significantly lower (P < .001) clinical respiratory scores when compared with unvaccinated pigs, but at study days 28 to 56. On farm C, vaccinated pigs had significantly lower (P = .002) clinical respiratory scores when compared with unvaccinated pigs at study days 21 to 63 (Figure 1).

Growth performance

The body weight of pigs at study day 0 (21 days of age, time of vaccination) did not differ significantly between the vaccinated and unvaccinated groups on all 3 farms. Vaccinated pigs from all farms (A-C) had significantly higher (P = .007 for farm A, P = .01 for farm B, and P = .03 for farm C) ADG at study days 0 to 49 (21-70 days old) when compared with unvaccinated pigs from the same farm. Additionally, farm C vaccinated pigs had a significantly higher (P = .031) ADG at study days 50 to 91 (71-112 days old) when compared with the unvaccinated pigs. Overall (study days 0-154), the difference between vaccinated and unvaccinated groups was significant (P = .02 for farm A, P = .02 for farm B, and P = .02 for farm C) on all 3 farms (Table 1).

Quantification of M hyopneumoniae in nasal swabs

On farm A, vaccinated pigs had a significantly lower (P = .009) number of genomic copies of M hyopneumoniae in their nasal swabs when compared with unvaccinated pigs at study day 21. On farm B, there was a numerical, but not statistically significant (P = .05), difference in the number of M hyopneumoniae genomic copies on the nasal swabs of vaccinated and unvaccinated pigs. Farm C vaccinated pigs had a significantly lower (P = .02 at study day 21, P = .03 at study day 49, and P = .001 at study day 77) number of M hyopneumoniae genomic copies in their nasal swabs when compared with unvaccinated pigs at study days 21, 49, and 77 (Figure 2).

Serology

On farm A, vaccinated pigs had a significantly higher M hyopneumoniae ELISA S:P ratio at study days 49 (P = .001) and 77 (P = .006) when compared with unvaccinated pigs. On farm B, vaccinated pigs had a significantly higher (P = .001) M hyopneumoniae ELISA S:P ratio at study days 21, 49, and 77 when compared with unvaccinated pigs. On farm C, vaccinated pigs had a significantly higher (P = .001) M hyopneumoniae ELISA S:P ratio at study days 49 and 77 when compared with unvaccinated pigs (Figure 3).

ELISpot

On farm A, vaccinated pigs had a significantly higher (P < .001) number of M hyopneumoniae-specific IFN-γ-SC at study day 49 in their PBMC when compared with the unvaccinated pigs. On farm B, vaccinated pigs had a significantly higher number of M hyopneumoniae-specific IFN-γ-SC in their PBMC at study days 21 (P = .01) and 49 (P = .001) when compared with the unvaccinated pigs. On farm C, vaccinated pigs had a significantly higher (P = .002) number of M hyopneumoniae-specific IFN-γ-SC at study days 49 and 77 in their PBMC when compared with the unvaccinated pigs (Figure 4).

Pathology

Vaccinated pigs had significantly lower (P < .001) macroscopic and microscopic lung lesion scores when compared with the unvaccinated pigs on the 3 farms at study day 154 (Table 2).

Discussion

In the present field trial, vaccination against M hyopneumoniae reduced the severity of lung lesions and clinical signs, including coughing, which resulted in improved growth performance. Controlling M hyopneumoniae and its associated diseases in the field can be challenging. Vaccination against M hyopneumoniae using commercial vaccines is the most common strategy within Asian swine production systems. The major advantages of vaccination include reduction of clinical signs and pneumonic lung lesions and improvement of daily weight gain in field trials.^17-20 No statistically significant difference was observed in the growth performance (ADG) over the nursery period between groups. This confirmed that vaccine did not have a detectable negative impact on growth performance shortly after injection. Overall (study days 0 to 154), the difference in growth performance between vaccinated and unvaccinated pigs was significant on all 3 farms where M hyopneumoniae was circulating.

The mycoplasma organism is a small bacterium without a cell wall. It is a unique pathogen in that it does not invade the body, but instead colonizes the mucosal surface of the respiratory tract damaging the cilia.^21,22 Therefore, the serum antibody response to the bacteria may be variable and not a great measurement of protective immunity. No correlation between vaccine-induced serum antibody levels and protection from colonization and disease has been determined.^13,23 Although protective immunity against M hyopneumoniae is not fully understood, cell-mediated immunity is likely to play an important role in the protection against M hyopneumoniae infection as described in previous studies.^13,23 In this study, M hyopneumoniae-specific IFN-γ-SC gradually increased from day 21 and reached a peak at day 49. During this period, vaccinated groups improved ADG and reduced respiratory signs significantly compared with unvaccinated groups on the 3 farms. These results indicate that M hyopneumoniae-specific IFN-γ-SC may provide protective immunity. However, since increased levels of IFN-γ-SC coincide with the increased amount of mycoplasmal loads in nasal shedding, further studies are needed to determine the functional role of cell-mediated immunity as a protective immunity.

The clinical impact of reducing nasal mycoplasmal shedding by vaccine may be controversial. The vaccine used in this study reduced the genomic copies of M hyopneumoniae on the nasal swabs from vaccinated pigs. Similarly, some studies indicate that other commercial vaccines may also reduce the number of organisms in the respiratory tract and may decrease the infection level in a herd.²⁴ Contradictory to these findings, additional field studies have shown that vaccination does not significantly reduce the transmission of this respiratory pathogen.²⁵ In addition, vaccines do not prevent colonization.^17-19,26 Consequently, vaccination alone will not be sufficient to eliminate M hyopneumoniae from infected pig herds. The producer must still pay attention to stocking density, ventilation, biosecurity, and the control of other diseases to be successful in the long-term control of mycoplasma.

Different sampling sites were used to detect M hyopneumoniae infection by PCR on experimentally and naturally infected pigs. Laryngeal swabs were a reliable sample for early detection of M hyopneumoniae, followed by broncho-alveolar lavage fluid and nasal swabs in live experimentally infected pigs, especially during the acute period.²⁷ In contrast, the most sensitive sampling sites in live naturally infected pigs were tracheo-bronchial swabbing and tracheo-bronchial washing, as compared to oral-pharyngeal brushing and nasal swabbing.²⁸ This may partly explain the relative inaccuracy of the nasal swabbing method.²⁸ In the present study, sterile swabs were inserted into nasal turbinates deeply and rotated hard enough on the inside of the nose to collect the samples properly for the detection of M hyopneumoniae. In addition, nasal swabs are practical samples for the detection of M hyopneumoniae under field conditions.

Mycoplasma hyopneumoniae is a slow-growing bacterial organism with a long period between infection and clinical impact.²⁹ Early infection during the life of a pig is important for the organism to grow and develop clinical disease in pigs. Mycoplasma hyopneumoniae prevalence at weaning can be an important indicator of disease severity in growing pigs.³⁰ Thus, control measures directed at lowering M hyopneumoniae prevalence at weaning could have a significant impact in disease presentation in grow-finishing pigs. This enhances the criticality that early control of M hyopneumoniae infection by vaccination is essential to control mycoplasma pneumonia. Early vaccination of piglets (< 3 weeks of age) is more common in single-site herds in Korea. Early vaccination has the advantage that immunity can be induced before the pigs become infected, and that fewer pathogens are present to possibly interfere with an immune response. In this field trial, commercial M hyopneumoniae vaccine was also administered to piglets at 3 weeks of age as recommended by company claims.

Single-dose M hyopneumoniae vaccination at 3 weeks of age significantly improved growth performance in pig farms suffering from M hyopneumoniae infection. This field trial was conducted on 3 farms and included housing conditions and a health status reflecting those of conventional facilities in Korea. The results of this study demonstrate that the newly introduced M hyopneumoniae vaccine provided good protection against M hyopneumoniae on farms.

Implications

Under the field conditions of this study:

• Mycoplasma hyopneumoniae bacterin effectively improved growth performance.
• Mycoplasma hyopneumoniae bacterin reduced pathological lung lesions.

Acknowledgments

This research was supported by contract research funds (Grant No.550-20180057) of the Research Institute for Veterinary Science (RIVS) from the College of Veterinary Medicine and by the BK 21 FOUR Future Veterinary Medicine Leading Education and Research Center (Grant No. A0449-20200100). The first two authors contributed equally to this work.

Conflict of interest

None reported.

Disclaimer

Scientific manuscripts published in the Journal of Swine Health and Production are peer reviewed. However, information on medications, feed, and management techniques may be specific to the research or commercial situation presented in the manuscript. It is the responsibility of the reader to use information responsibly and in accordance with the rules and regulations governing research or the practice of veterinary medicine in their country or region.

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