Cross-protection of a porcine circovirus types 2a/b (PCV-2a/b) and Mycoplasma hyopneumoniae trivalent vaccine against a dual PCV-2e and Mycoplasma hyopneumoniae challenge

Abstract

The purpose of this experimental study was to determine the cross-protection of a new trivalent vaccine containing porcine circovirus types 2a/b (PCV-2a/b) and Mycoplasma hyopneumoniae. Pigs were vaccinated intramuscularly at 21 days of age, then challenged at 42 days of age with a dual PCV-2e and M. hyopneumoniae challenge. Growth performance was significantly improved during the experimental period (21 to 63 days of age) in vaccinated-challenged pigs compared to unvaccinated-challenged pigs. Pigs that were vaccinated and challenged elicited a significant amount of PCV-2e- and M. hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SC) and reduced the levels of PCV-2e viremia and laryngeal shedding. The results of the present study demonstrated that a trivalent vaccine provided cross-protection against a dual PCV-2e and M. hyopneumoniae challenge.

Porcine circovirus type 2 (PCV-2), a small, closed-circular single-stranded DNA virus within the genus Circovirus and family Circoviridae is comprised of approximately 1800 base pairs (bp) and 2 major open reading frames (ORF) as ORF1 and ORF2 (1,2). Eight distinct PCV-2 genotypes, designated as PCV-2a to PCV-2h, have been identified to date, of which PCV-2a is the oldest (3). PCV-2 is the primary causative agent of porcine circovirus-associated disease (PCVAD) and is highly prevalent worldwide. PCVAD encompasses postweaning multisystemic wasting syndrome, porcine respiratory disease complex, reproductive disorders, and enteric diseases; all clinical manifestations of PCV-2 (4,5). Subclinical PCV-2 infection is currently the most common form of PCVAD that is spread worldwide (6). Another global pathogenic challenge for growing pigs is Mycoplasma hyopneumoniae, the causative agent of enzootic pneumonia (7). A chronic, dry, and non-productive cough, development of pneumonia with cranioventral consolidation, poor feed conversion efficiency, and poor growth performance are all characteristic of M. hyopneumoniae infection (8).

Pig samples geographically obtained from Mexico and the US in 2015 provided 4 new and distinct PCV-2 sequences (9). Upon analysis, 10 similar PCV-2 sequences (from 2006 onward) were also identified in the US in a retrospective analysis (10). The ORF2 of these newly discovered PCV-2 isolates presented only an 85% identity match to other known PCV-2 ORF2 sequences. They were 15 bp longer than that of PCV-2a or PCV-2b at 717 bp and 12 bp longer than that of PCV-2c and PCV-2d (9,10). As an 85% identity match of ORF2 sequences is considered substantially different from the previously recognized genotypes, the newly emerged genotype was labeled as PCV-2e (10). PCV-2e had since been detected in China (2017) followed by Korea in 2020 (11,12). PCV-2a, PCV-2b, and PCV-2d remain as the 3 major genotypes currently in field circulation (3), whereas PCV-2e is the most prevalent among all the minor genotypes (13).

Some of the largest worldwide economic losses in swine production are attributed to both PCV-2 and M. hyopneumoniae (14). Vaccination remains the most common and effective strategy in controlling infection from these 2 pathogens. Currently, Korean farms use combined vaccines containing PCV-2 and M. hyopneumoniae for more than 65% of their pigs (http://www.kahpa.or.kr). Most commercially available combined vaccines only contain one PCV-2 genotype in the formulation (15). As PCV-2e continues to emerge throughout major pig-rearing countries, vaccine efficacy concerns have risen over the current commercially available formulas (9–12). Recently, PCV-2e was isolated from the lymph nodes of a pig with PCVAD that had been vaccinated for PCV-2a with the rest of its herd (12). The objective of this study was to evaluate the efficacy of the new trivalent vaccine (PCV-2a/b + M. hyopneumoniae) in its ability to cross-protect against the newly emerged PCV-2e and M. hyopneumoniae under experimental conditions.

Eighteen, 14-day-old conventional, colostrum-fed pigs from sows that had never been vaccinated for PCV-2 were purchased from a high health commercial herd free of porcine reproductive and respiratory syndrome virus and M. hyopneumoniae but positive for PCV-2d, in which log₁₀ PCV-2d genomic copies/mL ranged from 2.23 to 3.12. These values were consistent with and met the definition to classify the herd for subclinical PCV-2d infection (16).

Pigs were tested upon arrival at the facility for this experimental study and determined to be seronegative against PRRSV (HerdChek PRRS X3 Ab test; IDEXX Laboratories, Westbrook, Maine, USA), PCV-2 (INgezim CIRCO IgG; Ingenasa, Madrid, Spain), and M. hyopneumoniae (M. hyo. Ab test; IDEXX Laboratories). Real-time polymerase chain reaction (PCR) was also performed on drawn samples, if pigs tested negative for PRRSV and PCV-2 (serum samples), and for M. hyopneumoniae (laryngeal swab) (17–19).

A total of 18 pigs were randomly distributed into 3 groups (6 pigs per group, n = 3 male and n = 3 female) and kept in different isolation rooms. All isolation rooms and pens were uniform in design and allowed pigs free access to feed and water troughs.

At −21 d post-challenge (dpc, 21 d of age), pigs in the Vac/Ch group received an intramuscular (neck) 2.0-mL dose of trivalent vaccine (Fostera^® Gold PCV MH, Serial No: 413369A, Expiration date: 03-Feb-2022; Zoetis). A 2.0-mL dose of phosphate-buffered saline (PBS, 0.01M, pH 7.4) was administered to each pig in the UnVac/Ch and UnVac/UnCh groups the same day.

At 0 dpc (42 d of age), pigs in the Vac/Ch and UnVac/Ch groups were inoculated with both PCV-2e (SNUVR199707, GenBank no. MN967003, 5th passage in PCV-free PK-15 cell lines) and M. hyopneumoniae (strain SNU98703). First, a 3-mL inoculation of PCV-2e challenge containing 1.2 × 10⁵ (50% tissue culture infective dose/mL) was administered intranasally. Five hours after PCV-2 inoculation, pigs were intramuscularly anesthetized with a mixture of 2.2 mg/kg xylazine hydrochloride (Rompun; Bayer Healthcare, Shawnee Mission, Kansas, USA), 2.2 mg/kg tiletamine hydrochloride and 2.2 mg/kg zolazepam hydrochloride (Zoletil 50, Virbac, Carros, France). A 7-mL dose of M. hyopneumoniae (strain SNU98703) culture medium containing 10⁷ color changing was intratracheally inoculated into the pigs (15,20).

At 21 dpc (63 d of age), all pigs were sedated by an intravenous injection of sodium pentobarbital and euthanized by electrocution as previously described (21). Tissues (lung, lymph node, tonsil, and intestines) were collected from each pig at necropsy, then fixed in a 10% neutral-buffered formalin solution, before they were embedded in paraffin. Blood and laryngeal swabs were collected from all pigs at −21, 0, 7, 14, and 21 dpc to perform PCR specific for PCV-2 and M. hyopneumoniae.

Animal observers were blinded to the type of challenge pathogens that were used for the duration of the project. Pigs were monitored daily for clinical signs and scored weekly using a score ranking system which ranged from 0 (normal), 1 (rough hair coat), 2 (mild dyspnea), 3 (moderate dyspnea), 4 (severe dyspnea and mild abdominal breathing), 5 (severe dyspnea and moderate abdominal breathing), and 6 (severe dyspnea and abdominal breathing) (22).

The live weight of each pig was measured at 21 (−21 dpc) and 63 (21 dpc) d of age. These values were applied to calculate average daily weight gain (ADWG; g/pig/d) between 21 and 63 d of age. These ADWG calculations were performed to analyze each of the production stages by calculating the difference between the starting and final weight divided by the duration of the stage. Pigs that either died or were removed from the study were still included in ADWG calculations.

Serum samples were collected for PCR analysis from pigs at −21, 0, 7, 14, and 21 dpc. DNA was first prepared through extraction from the collected samples with a commercial kit (QIAamp DNA Mini Kit; QIAGEN, Valencia, California, USA). PCV-2e genomic DNA copy numbers were then quantified by real-time PCR (19).

Laryngeal swab samples were also collected for PCR analysis at −21, 0, 7, 14, and 21 dpc. Prior to PCR analysis, DNA was first prepared through extraction from the collected samples with a commercial kit (QIAamp DNA Mini Kit; QIAGEN). Mycoplasma hyopneumoniae genomic DNA copies were then quantified by real-time PCR (17).

The collected serum samples were also tested with a commercial kit (INgezim CIRCO IgG; Ingenasa) for antibodies against PCV-2 and were considered positive if the optical density (OD) was > 0.3. The presence of M. hyopneumoniae antibodies was evaluated with another commercial kit (M. hyo. Ab test, IDEXX Laboratories) and samples were considered positive if the sample-to-positive (S/P) ratio was ≥ 0.4. Each of these criteria was set in accordance with the corresponding kit manufacturer’s instructions.

The amount of PCV-2e and amount of M. hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SC) present were measured with an enzyme-linked immunospot (ELISpot) assay. Peripheral blood mononuclear cells (PBMC) were stimulated using the aforementioned challenge strains for PCV-2e and M. hyopneumoniae (23,24).

The collected lung and lymphoid tissue sections were examined and scored by 2 blinded veterinary pathologists. Lung lesion severity scoring was based on peribronchiolar lymphoid tissue hyperplasia caused by mycoplasmal pneumonia lesions from 0 to 6 (25). Lymphoid lesion severity scoring was based on lymphoid depletion and granulomatous inflammation, marked and ranked on a scale from 0 to 5 (26).

A morphometric analysis of immunohistochemistry (IHC) and morphometric analysis of IHC were prepared by cutting 3 sections from each of 3 blocks of lung and lymph node tissue from each pig. The slides were analyzed using the NIH Image J 1.45s Program (http://imagej.nih.gov/ij/download.html) to obtain the quantitative data. PCV-2 analysis was conducted by randomly selecting 10 fields and the number of positive cells per unit area (0.95 mm²) was determined as previously described (27).

IBM SPSS Statistics for Windows version 23.0 (IBM, Armonk, New York, USA) was used to perform the statistical analyses for this study. Prior to analysis, all real-time PCR data were transformed to log₁₀ values and neutralizing antibody titers were transformed to log₂. The Shapiro-Wilk test was used to evaluate data for normal distribution. One-way analysis of variance (ANOVA) was used to examine differences in variables with normal distribution. Data from 1-way ANOVA tests that were statistically significant, were further evaluated by conducting a post-hoc test for a pairwise comparison with Tukey’s adjustment. Kruskal-Wallis test was used to examine differences in variables without normal distribution. Statistically significant Kruskal-Wallis test results were further evaluated with the Mann-Whitney test for a post-hoc test with Holm-Bonferroni adjustment. All results were reported in P-values; a value of P < 0.05 was significant.

Respiratory signs characterized by lethargy, coughing, and occasional sneezing with a marked decrease in appetite were observed in pigs in the UnVac/Ch group. Respiratory sign scores of pigs from the UnVac/Ch group at 14 and 21 dpc were significantly higher (P < 0.05) compared with those of pigs in the Vac/Ch and UnVac/UnCh groups.

A significant difference in average body weight ± standard deviation among the 3 groups was not present at the start of the experiment. Body weight was evaluated at 63 d of age (21 dpc). Pigs in the UnVac/Ch group weighed significantly less (P < 0.05) than pigs in the Vac/Ch and UnVac/UnCh groups. ADWG was compared between 21 and 63 d of age. Pigs in the UnVac/Ch group calculated as significantly lower (P < 0.05) compared with pigs in the Vac/Ch and UnVac/UnCh groups (Table I).

Table I.

Body weight, average daily weight gain (ADWG), and pathology data (mean ± standard deviation) among 3 groups at different days post-challenge (dpc).

	Age (dpc)	Vac/Ch	UnVac/Ch	UnVac/UnCh
Body weight (kg)	21 (−21)	5.47 ± 0.09	5.38 ± 0.15	5.48 ± 0.23
	63 (21)	16.92 ± 0.55a	15.57 ± 0.53b	17.55 ± 0.67a
ADWG (gram/days/pig)	21 to 63 (−21 to 21)	272.62 ± 14.85a	242.46 ± 11.29b	287.30 ± 15.22a
Lung lesion score	63 (21)	2.03 ± 0.50a	4.10 ± 0.65b	0.00 ± 0.00c
Lymphoid lesion score	63 (21)	1.33 ± 0.70a	3.03 ± 0.34b	0.00 ± 0.00c
Number of PCV-2-antigen positive cells	63 (21)	15.33 ± 3.63a	25.00 ± 6.64b	0.00 ± 0.00c

Different superscripts (a, b, and c) indicate significant (P < 0.05) difference among 3 groups.

ASWG — Average daily weight gain.

Significantly lower (P < 0.05) PCV-2e blood viral loads were measured in vaccinated-challenged pigs (Vac/Ch group) than in that of unvaccinated-challenged pigs (UnVac/Ch group) at 7, 14, and 21 dpc (Figure 1 A).

Figure 1.

Quantitation of porcine circovirus type 2e (PCV-2e) and Mycoplasma hyopneumoniae. A — Mean values of the genomic copy number of PCV-2e DNA in serum. B — Mean values of the genomic copy number of M. hyopneumoniae DNA in larynx from Vac/Ch (●), UnVac/Ch (○) and UnVac/UnCh ( ) groups. Variation is expressed as the standard deviation. Different superscripts (a, b, and c) indicate significant (P < 0.05) difference among 3 groups.

A significantly lower (P < 0.05) load amount of M. hyopneumoniae was detected in the larynx of vaccinated-challenged pigs (Vac/Ch group) than in that of unvaccinated-challenged pigs (UnVac/Ch group) at 14 and 21 dpc (Figure 1 B).

Pigs in the Vac/Ch group had a significantly higher (P < 0.05) PCV-2 S/P ratio (Figure 2 A) and PCV-2e-specific IFN-γ-SC levels (Figure 2 B) during the study when compared to the UnVac/Ch group at 14 and 21 dpc.

Figure 2.

Immune responses against porcine circovirus type 2e (PCV-2e) and Mycoplasma hyopneumoniae. A — PCV-2-specific ELISA S/P ratio. B — PCV-2e-specific interferon-γ secreting cells (IFN-γ-SC)/10⁶ peripheral blood mononuclear cells (PBMC). C — M. hyopneumoniae-specific ELISA S/P ratio. D — M. hyopneumoniae-specific IFN-γ-SC/106 PBMC from Vac/Ch (●), UnVac/Ch (○) and UnVac/UnCh ( ) groups. Variation is expressed as the standard deviation. Different superscripts (a, b, and c) indicate significant (P < 0.05) difference among 3 groups.

Pigs in the Vac/Ch group had a significantly higher (P < 0.05) M. hyopneumoniae S/P ratio than in the UnVac/Ch group at 7, 14, and 21 dpc (Figure 2 C). Pigs in the Vac/Ch group had a significantly higher (P < 0.05) M. hyopneumoniae-specific IFN-γ-SC level (Figure 2 D) than that in the UnVac/Ch group at 21 dpc (Figure 2 D).

Pigs in the Vac/Ch group had significantly lower (P < 0.05) microscopic lung and lymphoid lesion scores than the UnVac/Ch group at 21 dpc (Table I). Significantly lower (P < 0.05) numbers of PCV-2-antigen positive cells were measured in lymph nodes from vaccinated-challenged pigs (Vac/Ch group) (Figure 3 A) compared with the unvaccinated-challenged pigs (UnVac/Ch group) (Figure 3 B) at 21 dpc (Table I).

Figure 3.

Immunohistochemistry (IHC) for detecting porcine circovirus type 2 (PCV-2) antigens in lymph node. A — Representative IHC for detecting PCV-2 antigen (arrows) in lymph node from vaccinated and challenged with PCV-2e and Mycoplasma hyopneumoniae (Vac/Ch) group. Bar = 55 μm. B — Representative IHC for detecting PCV-2 antigen (arrows) in lymph node from the unvaccinated and challenged with PCV-2e and Mycoplasma hyopneumoniae (UnVac/Ch) group. Bar = 55 μm.

This efficacy study confirms that a PCV-2a/b and M. hyopneumoniae trivalent vaccine cross-protected pigs against a dual experimental challenge with PCV-2e and M. hyoneumoniae. PCV-2e infection alone rarely produces the typical lesions associated with PCVAD. Lesions are, however, more likely to develop if M. hyopneumoniae is inoculated at the same time (25). This is consistent with naturally occurring clinical PCVAD field cases, as they traditionally appear as a dual infection with PCV-2 and at least one additional pathogen(s). For these reasons, the dual challenge model is the appropriate choice for evaluating vaccine efficacy of PCV-2.

Growth performance evaluation was defined as the critical index of this study, as growth retardation is the common denominator of PCV-2 and M. hyopneumoniae infection. Statistical differences in growth performance were observed between the vaccinated-challenged and unvaccinated-challenged groups. These results are consistent with previous findings, in which vaccination of pigs with the same vaccine improved growth performance against dual PCV-2d and M. hyopneumoniae challenge (15).

This successful induction of significant amounts of IFN-γ is an important index of vaccine protection, as IFN-γ reportedly plays a critical role in the control of M. hyopneumoniae and PCV-2 infection (28,29). Vaccination with the study trivalent elicited PCV-2e-specific IFN-γ-SC. This in-turn reduced PCV-2e blood and lymphoid viral load, and reduced the severity of PCV-2e-associated lymphoid lesions. The same vaccines simultaneously elicited M. hyopneumoniae-specific IFN-γ-SC, that lead to a reduction in the levels of laryngeal load and a reduction in lung lesion severity. The trivalent vaccine used in this experiment protected pigs against a dual PCV-2e and M. hyopneumoniae challenge based on immunological, microbiological, and pathological analyses.

To our knowledge, this is the first report that evaluated the cross-protection of commercially available PCV-2a/b and M. hyopneumoniae trivalent vaccine against PCV-2e and M. hyopneumoniae. This trivalent vaccine induced a PCV-2e specific humoral immune response and pathogenic specific IFN-γ secreting cells, which provided cross-protection against PCV-2e. The PCV-2e strain used in this study was isolated from a 10-week-old, previously PCV-2a-vaccinated pig. This is consistent with vaccine failure within that PCV-2a-vaccinated herd. Commercial PCV-2 vaccines differ in adjuvant, formulations, and antigens. Antigens exist in many different forms such as inactivated whole virus, baculoviral expressed recombinant virus, and inactivated chimeric PCV-1/2 virus (30). Although the evaluated trivalent vaccine containing PCV-2a/b and M. hyopneumoniae provided cross-protection against PCV-2e in this study, these vaccine (especially antigen) differences listed should be considered; it cannot be assumed that all commercially available PCV-2a- and PCV-2b-based combined vaccines protect in the same manner.

Funding Statement

This research was supported by contract research funds (Grant no. 550-20190068) 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).