- Top of page
- Materials and methods
In livestock species, lifetime productivity and health may be limited by key events occurring during early embryonic and fetal development. As in humans, intrauterine growth restriction alters post-natal stress response, adipose tissue deposition, muscle development, glucose tolerance, blood pressure, catecholamine response, and the development of all vital organs including liver, spleen, heart, lung, muscle, and thymus.[1-3] Human epidemiological research also suggests that in utero growth restriction, associated with maternal undernutrition, may adversely affect humoral and cell-medicated immune responses in adolescence and adulthood. A limited number of studies of full-term, small-for-gestational-age babies in developing countries have demonstrated altered humoral and cell-medicated immune responses resulting in lower likelihood of response to Salmonella typhi vaccination, decreased thymopoeitin production (a marker for thymic growth and development), and higher risk of infection-related death.
In swine, the effects of pre-natal maternal stress on post-natal immunity has been most extensively studied using either daily restraint, rough handling, or the administration of adrenocorticotropic hormone (ACTH) during mid-gestation or late gestation. Maternal restraint during late gestation had an immunosuppressive effect on lymphocyte proliferation in response to various mitogens at 1 and 35 days of age, in addition to increasing piglet morbidity and mortality during the lactation period. The same animal model has demonstrated reduced tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and serum amyloid A production following LPS infusion in 5-week-old progeny after maternal restraint stress. Moreover, eight-week-old pigs derived from ACTH-treated dams demonstrated higher basal cortisol levels and slower healing times. Additionally, rough handling of dams, not the ACTH treatment, resulted in reduced sickness behavior in these 8-week-old pigs following LPS administration.
Low birth weight and high birth order are well known risk factors for reduced passive immunity and survival in pigs.[11, 12] While parity of birth may affect adaptive immunity, piglets born to gilts have reduced humoral response following vaccination; direct evidence for the impacts of birth weight on adaptive immunity and clinical disease severity is lacking and of great relevance to the modern swine industry. To compare severity between piglets born from high-birth-weight (HBW) and low-birth-weight (LBW) litters, swine influenza virus (SIV) infection was used as an exemplifier of neonatal respiratory disease in this study.
The objectives of this study were (1) to compare clinical, immunological, and pathological outcomes of influenza infection in HBW to LBW pigs and (2) to establish standardized sampling sites, score each site independently with set criteria, and compare scores between sites.
- Top of page
- Materials and methods
This experiment provides clear, albeit unexpected, insight into the impact of litter birth weight on the severity of swine influenza infection. Based on the research indicating that low birth weight is a risk factor for nosocomial influenza A infections, our hypothesis was that the severity of influenza infection in the LBW pigs would be more severe than in the HBW pigs. The opposite occurred, in which severity was lower in LBW compared with HBW piglets. Significant differences, however, were limited to pathology and only in specific lobes. Cytokine protein production, virus shedding, and clinical respiratory signs did not differ between groups. We suspect that this is partly due to the relatively low experimental power (0·32–0·68 depending on outcome variable) and the considerable between-pig variation. To demonstrate statistical differences across other variables, a similar future experiment would need sample sizes of 40–50 litters (selecting 4 piglets per litter) or 60–100 litters (selecting 2 piglets per litter) based on the group means and variances generated in the present study.
There are many reasons why a sow may deliver a low- or high-birth-weight litter, and some sows flip-flop delivering high- and low-birth-weight litters in successive parities. However, a small proportion of sows consistently deliver high- or low-birth-weight litters. Ongoing research conducted by our group has demonstrated that the litter birth-weight phenotype is not fully expressed until sows have repeatedly delivered multiple consecutive high- or low-birth-weight litters. Although evaluating the progeny from these repeatable sows is our overarching research goal, in this study, we were unfortunately not able to include progeny from sows older than second parity. So although litter birth weight and Z-score differed significantly by group, we were unable to specifically select progeny from dams delivering repeatable phenotypes. This may or may not have impacted our results. The exact mechanisms related to the delivery of repeatable HBW or LBW phenotypes are not fully understood at this time, but it may involve epigenetic programming, and we have studies underway to investigate this phenomenon.
A number of studies have linked influenza susceptibility to low- birth-weight and epigenetic programming. Firstly, it is proposed that the explosive mortality associated with the 1918 H1N1 pandemic may be associated with acquired epigenetically mediated immunological differences among birth cohorts in combination with the emergence of a new strain/subtype. It is difficult to prove whether or not this theory is valid; however, epigenetic programming has been linked to the metabolic syndrome (hypertension, insulin resistance, dyslipidemia, obesity), and obesity indirectly increases the susceptibility to a number of community-borne pathogens, including influenza, through number of potentially altered immunological mechanisms.
Direct epigenetic links to respiratory health have also been reported. Pre-natal and early childhood environmental programming is directly associated with the risk of asthma and allergic airway disease.[30, 31] Moreover, host defenses in response to influenza infection involve a number of antiviral mechanisms including pro-inflammatory interleukin-32 (IL-32), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). It has been demonstrated that epigenetic changes that upregulate the DNA methyltransferase (DNMT) enzymes act to silence genes and decrease expression of IL-32, COX-2, and PGE2, thus reducing host defenses and viral clearance.[32, 33] In theory, epigenetic programming events that alter the methylation of genes responsible for pro-inflammatory or other immunological pathways may alter susceptibility or severity of influenza in humans and possibly in animals. Why influenza severity was greater in HBW pigs in the present study is unclear and warrants further investigation. While it is possible that our findings are spurious, it is also possible that mechanisms affecting influenza susceptibility in pigs differ from those of humans.
In spite of the fact that the birth weights differed by group by approximately 0·3 kg, weaning weight did not differ statistically. This is not surprising because there are numerous factors affecting average daily gain (ADG) during the lactation period including litter size, parity of dam, sow feed intake, cross-fostering procedures, and room temperature. Piglets selected for this study suckled their birth sow and were of average weight at birth compared with their siblings. Although group differences in weaning weight were identical to the differences in birth weights (0·3 kg), there was considerably more variation in weaning weight than in birth weight, resulting in the lack of statistical significance. To evaluate whether lactational ADG was related to influenza severity, it was tested in all statistical models as a potential predictor variable, but was found to be non-significant in all. Although excessive childhood weight gain following fetal and infant malnourishment is a risk factor for metabolic syndrome in humans, it was not significantly associated with influenza severity in the present study.
A limitation of this study was the lack of a non-inoculated reference control group. Although the primary objective of the study was to compare high- and low-birth-weight pigs, the addition of a reference control group would have primarily served to verify whether the source farm remained free of swine influenza as well as the absence of pneumonic lesions and BALF cytokines in non-infected pigs. The experiment used small batches of pigs over a number of months, and each successive batch tested negative for swine influenza by serology a week before arrival and by PCR at arrival. We are confident that the source farm remained negative throughout the experiment due to the absence of respiratory disease in the source farm and lack of seroconversion. Moreover, the negative control BALF was collected from a pig that came from the same source farm and was kept in the BSL2 facility for 4 weeks. This animal was SIV, PRRSv, PCV2, and Mhyo negative at necropsy.
Another interesting finding in this study involved the morphometric techniques that we applied to limit variation in sampling and scoring methods. Using systematic sampling of the lung lobes at 2·5 cm from the lobar tip regardless of lesions seen was especially helpful in this study because we were sampling the animals at 48 hours after inoculation (3 days earlier than is customary). For several pigs, there were minimal to no gross lesions seen in the lungs, but these animals had both microscopic lesions and corresponding influenza immunoreactivity. This is a conundrum faced in experimental studies when treatments are compared, and there are minimal to no lesions seen at necropsy. In most studies, the investigator often collects one or two samples containing a lesion, and these sites vary from animal to animal. For the lungs that have no lesions, a pre-established sample, such as a section from the right middle lobe, may be collected. In an attempt to standardize the sampling process for influenza studies, specific sites were selected for this study.
The sampling sites for this study were chosen based on the personal observations by the pathologist (SED) on over 250 necropsies of experimentally inoculated pigs (both intratracheal and intranasal) where the majority of lesions spanned the area approximately 2–3 cm from the tip of each cranial and middle lobe.[19, 35, 36] As a pilot study, the sampling method and scoring were also compared for 60 of these pigs (comparison not published).
Using the individual lung lobes and summative scores, the findings of significant differences were variable in location, but most consistent in the right middle lung lobe where both the microscopic and immunohistochemistry scores were significantly different between LBW and HBW groups. However, when the microscopic scores were compared between the lobes and within the birth weight groups, the right cranial lobe was significantly different. This finding is most likely related to the fact that pigs have a tracheal bronchus that enters the right cranial lung lobe and is cranial to the carina. This anatomical variation in pigs could allow a higher concentration of the inoculum to enter the right cranial lung lobe than the other lobes. It also points to the fact that it is important to make sure that the animals are consistently placed in the same position for waking from the anesthetic after being inoculated, so that the gravitational pull on inoculum in the airways is similar between pigs.
Another reason for standardizing the tissue collection procedures is to enable the use of more advanced applications in our analysis. In general, the microscopic and immunohistochemistry scores are assigned by pathologists that are blinded to the treatment groups. However, there is always a certain degree of subjectivity to these scores. If we apply morphometric scoring techniques, such as using a computer program to measure the color intensity from microphotographs of an immunohistochemistry slide, then we have a quantitative, instead of a qualitative score for the statistical analysis.
In summary, there were two key findings in this study. Using the SIV infection model, we found that HBW pigs had more severe lesions than LBW pigs when infected with TX98 for 48 hours. The results of this study also demonstrated a standardized method that could be applied in future studies where morphometric scoring techniques are used. However, further assessment is needed to determine the repeatability and accuracy of the proposed sampling and assessment methods for influenza studies.