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2011, Vol. 6 No. 2, Article 90

 

Evaluation of Innate Immunity in Pigs
Under Field Conditions

Livia Moscati, Marco Sensi, Lorenzo Battistacci, Ivonne Laura Archetti and Massimo Amadori *1

 

Laboratory of Clinical Sciences,
Istituto Zooprofilattico Sperimentale Umbria e Marche,
via G. Salvemini 1, 06126 Perugia, Italy

1Laboratory of Cellular Immunology,
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna (IZSLER),
via A. Bianchi 9, 25124 Brescia, Italy

 

 

*Corresponding Author; e-mail address: massimo.amadori@izsler.it

 


ABSTRACT

The objective of this study was to investigate some innate immunity parameters of pigs under different environmental and clinical conditions. In a preliminary phase, physiological values of serum lysozyme, haemolytic complement (classical pathway), bactericidal activity and haptoglobin were assessed in healthy pigs of different ages to define a range of reference values. Then, the same assays were employed in “problem” pig farms, characterized by repeated occurrence of opportunistic disease outbreaks. Under poor environmental conditions the levels of the parameters under study were shown to be significantly different from the above range prior to, as well as following disease outbreaks. Also, the above range of values was observed in the same ”problem” farms after major improvements of the housing conditions. Results indicate that a combined scheme of clinical and environmental inspections associated with a timely assessment of clinical immunology parameters can be a practical and accurate reporter system of pigs’ environmental adaptation and a suitable readout of interventions for better health and welfare conditions.

KEY WORDS

Swine, innate immunity, disease, adaptation, animal welfare.

INTRODUCTION

Various environmental effects may adversely bear on welfare of pigs under intensive farming conditions, often leading to chronic rather than acute stress as a long-term outcome of modern husbandry techniques. Thus, long-lasting homeostatic control actions are often required vis-à-vis unfavourable climate and microclimate conditions (temperature, humidity, draught), microbial infectious pressure, pain, fear, inability to perform a defined behavioural repertoire, barren environments, boredom, inadequate diet, metabolic stress for meat production, improper farmer’s behaviour (stockman’s effect). Also, genetic selection for lean pigs has caused the appearance of some undesirable traits, which are likely to worsen the adaptation process to modern husbandry techniques. For example, the percent weight of the heart muscle has decreased from 0.38% in wild boars to 0.21% in modern Landrace pigs (Brambilla et al., 2002).
Regardless of the actual conditions, individuals are prompted to adapt in order to improve and optimize the interaction with their environment. In this respect, animals usually adopt a “feed forward” strategy: animals mount a corrective action to potentially noxious stimuli before any problem becomes substantial. This implies a stepwise corrective action, whereby activity and energy expense are proportional to the perceived threat (Broom, 2006). Welfare is thus the result of the adaptation process, including adaptation to disease agents: in this case animal welfare is equal to animal health (Broom et al., 2006). In general, whenever animals are forced to severe, prolonged coping reactions with a considerable energy expense, welfare is poor and a serious depression of the immune system eventually turns out as one of the negative outcomes (Amadori et al., 2009). In addition, a conflict often arises in farmed pigs between immune response and performance under conditions of heavy infectious pressure, as previously shown in the M. hyopneumoniae model (Pointon et al., 1985). In this scenario, reduced performance often ensues as a result of abnormal inflammatory cytokine responses (Harding et al., 1997).
Environmental adaptation of pigs may be of concern in several farms where lean pig phenotypes, their pressing needs and inability to cope with sub-optimal environmental conditions are confronted with poor management and infrastructure. As a result, swine practitioners need to carry out a timely assessment of environmental adaptation and to recognize possible risks of disease occurrence. In addition, the measures adopted for improving housing and management conditions of pigs should be checked by a proper readout system. For this purpose, innate immunity parameters may be of some importance. They are affected by environmental conditions and are predictive of opportunistic disease occurrence, as previously shown in a cattle model (Amadori et al., 1997). Some innate immunity parameters in pigs (serum lysozyme, haptoglobin, complement and bactericidal activity) were extensively dealt with in the past (Taylor, 1983; Hill and Porter, 1974; Schulze and Muller, 1980; Linscott, 1986; Petersen et al., 2004). In addition, clinical signs of lameness, respiratory disease, diarrhoea, tail bite and ear necrosis are reflected in high haptoglobin concentrations in swine sera (Petersen et al., 2002). Therefore, we reasoned that such innate immunity parameters could characterize the pig’s adaptation process and the related clinical outcome. To test this hypothesis, we set out to validate robust, user-friendly formats of the relevant clinical immunology tests. These were applied first on healthy pigs of different age groups and then in “problem” herds, in which the immune parameters under study were investigated before, during and after outbreaks of opportunistic diseases.

MATERIALS AND METHODS

Animals Our study was conducted on healthy and diseased pigs under the regular veterinary practices approved by the Italian veterinary practitioner associations, under the supervision of the Animal Health and Welfare Division of the Italian National Veterinary Services.
Serum lysozyme
Serum lysozyme was assessed by the lyso-plate assay (Osserman and Lawlor, 1966). Serum samples were reacted with a suspension of Micrococcus lysodeikticus in agar gel in 10-cm Petri dishes. Serum samples were distributed in duplicate in 3-mm holes, 2 cm apart, at a regular distance of 1.5 cm from the dish edge. Contrary to the original protocol, the reaction was carried out at 37°C, 18 h, in a humidified incubator. The diameter of the lysis areas around serum samples and lysozyme standards of known concentration in phosphate buffer 0.066 M pH 6.3 was assessed by calibers or rules. Under these conditions, lysozyme concentration (μg/ml) is proportional to the diameter of lysis areas and is determined from a standard curve created with reference preparations of egg white lysozyme (Sigma-Aldrich, St Louis USA).
Total Haemolytic Complement
Total Haemolytic Complement (classical pathway) was assessed following the procedure described by Seyfarth, (1976), using a rabbit haemolysin/sheep erythrocytes haemolytic system. The volumes of the reagents were modified to perform the test in microtitre plates at a final volume of 125 ml/well (100 ml of serum dilutions + 25 ml of 3% haemolytic system). 0 and 100% haemolysis controls were set up in each plate at the same volume in Veronal buffer pH 7.3 and distilled water, respectively. Titres were expressed as 50% Haemolytic Units (C’H50) / 100 ml (the test volume of sera). Reference standard sera stored at –80°C were used in case to correct the values of haemolysis for the different batches of sheep erythrocytes.
Serum bactericidal activity (SBA)
The technique was earlier used as a plate count assay (Dorn et al., 1980) and was later adapted for convenience for a turbidometric assay in microtitre format (Amadori et al., 1997). Briefly, E. coli non-pathogenic Perugia strain, negative for expression of K88 antigen, Verotoxin, heat-stable and heat-labile toxins, was grown till log phase in 20 ml of Brain Heart Infusion broth (BHI, Biolife Italiana, Milan, Italy) and frozen at –80°C in sterile skim milk. For each test one aliquot was thawed, resuspended in 15 ml of BHI medium and incubated at 37°C till OD 590nm was doubled. Then, bacteria were diluted 1:100 in sterile saline. Test reagents were distributed into wells of sterile U-bottomed microtitre plates as follows: 50 ml of test serum (in duplicate) + 50 ml of Veronal buffer pH 7.3 + 100 ml of BHI broth + 10 ml of 1:100-diluted bacterial suspension. Controls of sterility were set up without bacteria. Controls of bacterial growth (growth control) were set up without serum. The missing components were replaced by Veronal buffer pH 7.3 at the same volumes. Plates were incubated in a humidified box at 37°C for 18 hours. Then, they were read spectrophotometrically in an ELISA reader at 690 nm, with blank set on the sterility control.
% SBA was derived from the following formula:
% SBA = 100 - (OD Test Sample x 100/OD growth control)

Haptoglobin
Haptoglobin was measured by using the commercial kit (Phase Haptoglobin Colorimetric Assay, Tridelta Development Ltd, Kildare, Ireland).
Clinical chemistry
Total protein, iron and zinc in serum samples were assessed by a Konelab 2001 biochemical analyzer using specific kits (Sentinel Diagnostics, Milan, Italy). Albumin, α, β, and γ-globulins were measured by electrophoresis on cellulose acetate strips (Theoria 2000 M apparatus, Biogroup Medical System, Talamello, Italy), followed by staining of protein bands with a specific kit (Biodevice, Rimini, Italy) according to the manufacturer’s directions, and densitometric examination on a Digiscan apparatus (Crony Instruments, Rome, Italy).
Antibody Tests
The following test kits were used according to the manufacturer’s directions:
• Pseudorabies virus (PRV): HerdChek Pseudorabies Virus gpI ELISA test kit (IDEXX Laboratories Inc., Westbrook, Maine)
Mycoplasma hyopneumonie: HerdChek Mycoplasma hyopneumoniae Antibody Test kit (IDEXX Laboratories Inc., Westbrook, Maine)
Virus isolation
Swine Influenza Virus (SIV) infection was investigated by intrallantoic inoculation of tissue homogenates in embryonated hen eggs at days 9-11 of incubation. Six days later, embryonated eggs were cooled at 4°C and an agglutination test of chicken red blood cells was carried out on a sample of allantoic fluid.
TaqManÒ probe Real-Time PCR
This was adopted for detection of Porcine Respiratory and Reproductive Syndrome virus (PRRSV) and Porcine Circovirus 2 (PCV2) in serum samples. The reaction was performed in the CFX96TM Real-Time System (Bio-Rad, Milan, Italy).
The PRRS virus amplification was performed by one-step reaction using primer set and probe designed on ORF6 gene of PRRS virus, as described by Revilla-Fernandez et al., (2005). Each 20 ml PCR mixture consisted of 1× QuantiTect Virus Master Mix, 1×QuantiTect Virus RT Mix (Qiagen, Milan, Italy), 0.4 mM of each primer, 0.2 mM of probe and 3 ml of total RNA. The amplifications conditions were: RNA retrotranscription at 50ºC for 20 min, then 95ºC for 5 min followed by 40 cycles of denaturation at 95ºC for 15 s, annealing-extension at 60ºC for 45 s and plate reading (acquisition of fluorescent data). The fluorescence threshold limit of the CFX96TM Real-Time System was set automatically.
PCV2 amplification was performed using primer set and probe designed on Cap gene of PCV2 virus, on the basis of a previous study (Chung et al., 2005). Each 20 ml PCR mixture consisted of 1× QuantiTect Virus Master Mix, (Qiagen, Milan, Italy), 0.4 mM of each primer, 0.2 mM of probe and 3 ml of genomic DNA. The amplification conditions were: 95ºC for 5 min followed by 40 cycles of denaturation at 95ºC for 15 s, annealing-extension at 60ºC for 45 s and plate reading (acquisition of fluorescent data). The fluorescence threshold limit of the CFX96TM Real-Time System was set automatically.
Healthy animals
In the first phase of this study, values of serum lysozyme, complement, bactericidal activity and haptoglobin in swine were reviewed in the available publications and also in an Italian data base on the web (http://www.izsler.it/izs_bs/ftp//doc/CREF Benessere20animale/Attivita/Immunologia_clinica/Biochimica1.pdf).
Commercial farms were selected on the basis of their excellent production and animal replacement figures, and the low incidence of disease. In addition, all the samples were collected from pigs showing satisfactory health and growth, housed under good hygiene conditions.
A study was carried out over two years in a commercial farrow-to-finish operation, in which the above parameters of innate immunity were monitored in different age groups (Table 1). The herd was classified as "normal" on the basis of the clinical picture, the good housing and management conditions, as well as the farm average production figures at the beginning of the trial:
• Number of litters per year: 2.3
• Number of piglets born alive/farrow: 12.2
• Litter size at weaning: 11.3
• Piglets weaned per sow/year: 26.1
A total of 1,440 serum samples in two years were tested in this study for lysozyme, complement, bactericidal activity. Samples were collected from sows and piglets during the main phases of the production cycle (60 sows controlled for 4 productive cycles and 120 piglets monitored 3 times, during the pre-weaning, fattening and finishing cycles). Samples from sows were balanced for parity order. All samples were collected in the morning before feeding.
Problem herds: trial 1
A trial was conducted on farrow-to-finish herd with 250 sows from Dutch genealogy book that had repeatedly shown diverse disease problems during the pre-fattening phase, up to the age of 70 days. A total of 100 blood samples in vacuum tubes were randomly collected at five subsequent intervals (20 samples at a time) from 1, 15, 30 to 40, 50 to 60, 80 to 90-day old pigs of the same group. Immunological assays and antibody tests for Pseudorabies virus (PRV) and Mycoplasma hyopneumoniae infections, as well as virological and PCR tests for Swine Influenza Virus (SIV), Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) and Porcine Circovirus-2 (PCV2) were carried out on each serum sample. Environmental swabs for total mesophilic aerobic bacterial counts (Colony Forming Units, CFU) were collected from floor, hoppers, nests of the farrowing crates and plastic bars on the grid floor of the weaning cages.
Problem herds: trial 2
The trial was carried out in a farrow-to-finish herd with 450 sows (British hybrids), where an hyper-acute respiratory syndrome with high morbidity and mortality was reported in the growers’ unit (mean weight > 40 Kg ). At the early onset of the reported syndrome animals showed weakness, staggering gait, anorexia, body temperature over 41°C, shallow breathing. Three of the most severely affected pigs were slaughtered; post mortem findings and laboratory investigations led to the diagnosis of hyper-acute pleuro-pneumonia sustained by Actinobacillus pleuropneumoniae. Blood samples were collected from twenty, randomly selected animals of the affected group every 15 days for three times in a row to check the innate immunity parameters under study.
Problem herds: trial 3
Innate immunity parameters were investigated in a breeding unit with 200 sows from Dutch genealogy book that had suffered high newborn mortality (> 20%), mostly in 1 to 3-day old piglets in farrowing rooms with slatted-floor crates above the ground. In the farm under study both ventilation and temperature were computer-controlled. Thermal measures with probes at ground level under the crates showed inadequate control of temperatures, which fluctuated between 11º and 16º C over one week in November. A thorough inspection of the affected pens revealed sows with body condition scores (BCS) below the expected values in both gestation and farrowing rooms, wide weight differences in newborns associated with long dull hair coat, lack of nests and pads, diarrhoea in suckling piglets of different ages with a higher incidence in the neonates, high prevalence of runt piglets.
Problem herds: trial 4
The trial was carried out in winter on pigs from Danish genealogy book in two fattening farms. Farm 1 (“problem” herd) consisted of two pens housing weaner and growing-finishing pigs, respectively. Pigs were introduced at 3 months of age and live weight of pigs at slaughter (10-11 months of age) amounted to 140 – 170 Kg. The growing-finishing unit had concrete flooring with a small grid area (about 50 cm-wide) in the lateral zone of each box, near the drinkers. Pressurized water was daily used for cleaning of boxes. Natural venting was operated by guillotine-like windows in the presence of a double-slope roof. A corridor along the main wall provided access to each box. In the second pen, weaners were housed for 30-40 days before entering the growing-finishing unit. Because of the frequent arrivals of new animal consignments of various origins, no all-in-all-out housing scheme was being applied during the trial. Disease cases sustained by Actinobacillus pleuropneumoniae had been present for a long time with high morbidity and mortality. “Problem” herd 1 was compared with herd 2, located in the same area and consisting of two fattening pens for a total of 1,500 pigs. These were born in the same breeding unit, moved to herd 2 at a weight of some 30 Kg (70-80 days of age) and further reared up to a slaughter weight of 160-170 Kg. The double-slope roof was equipped with a central longitudinal cupola along the entire length of the pen. Grid flooring covered the whole surface of the boxes. An all-in-all-out housing scheme was routinely applied. Liquid feed was administered in troughs, each serving two adjacent boxes. The overall clinical conditions of pigs were satisfactory. Owing to the above, it was decided to compare the time-course of clinical immunology and clinical chemistry parameters in the two herds with such a different sanitary and management status. for this purpose, farms were inspected and blood samples from 15, randomly chosen animals of the same age were collected in “problem” herd 1 and herd 2 at monthly intervals.
Statistical analyses
One-way ANOVA and subsequent Newman-Keuls Multiple Comparison Test were applied to the data obtained in trials 1 and 2. Two-way ANOVA was applied instead to the data sets of trial 3 and 4. The differences between the 2 herds under study in trial 4 were further checked by a t test for each immunity parameter and sampling time (Prism 2.01, GraphPad Software, San Diego, CA).

RESULTS

Healthy animals
By a proper combination of the WEB data base and the field study, the two sets of data were shown to overlap to a great extent. As a result, the following reference values were adopted as thresholds of acceptance in pigs from the pre-weaning to early fattening periods (120-day old animals), as well as in sows and gilts:
1. Lysozyme: 1 – 3 μg / ml
2. Total haemolytic complement (classical pathway): 50 C’H50 / 100 ml
3. Serum bactericidal activity: 40%
The above levels represented on the whole the 80th percentile of the values obtained in our field trial on healthy, outbred pigs of the aforementioned age groups and categories in a farm with very low levels of environmental stressors. Also, possible subclinical infections in the animals under study were unlikely because of the favourable farm production figures.
In addition, reference values of 0.2 – 2.0 mg/ml for swine haptoglobin were adopted on the basis of the enclosed directions for use in the aforementioned kit. This range of values was later confirmed on all the sera of healthy pigs examined to date in our lab (Moscati et al., unpublished data).
Serum values above those reported for lysozyme and haptoglobin and below those of complement and SBA were considered as outcome of poor environmental adaptation and possible predictors of disease occurrence.
Problem herds: trial 1
Serological, virological and PCR tests did not show the presence of infections by common swine pathogens. On the contrary, clinical immunology tests revealed a profound alteration of innate immunity. In fact, a significant decrease of serum bactericidal activity took place in the pigs under study (P<0.001, Table 2). Under these conditions, the use of feedstuff supplemented with wide-spectrum antibiotics helped prevent a disease condition in the weaning cages. Nevertheless, in the 30 to 40 and 50 to 60-day old pig groups the serum bactericidal activity values were much below the standard values, while serum lysozyme and haemolytic complement values were close to the normal range. When animals were moved to the pre-fattening rooms (without an “all in/all out” procedure), the unfavourable conditions caused the occurrence of disease cases. In fact, one week after the introduction into the pre-fattening rooms the growers under study experienced a progressive reduction of the growth rate, respiratory symptoms, paleness, weakness; a high percentage died or became runts in a month’s time. Post-Weaning Multisystemic Wasting Syndrome (PMWS) and PRRS were suspected even though serum samples tested negative for PCV2 and PRRSV genomes in Real-time PCR analyses. Surface swabs in the pigs’ pens revealed a high microbial load (3,000,000 – 9,500,000 CFU / 10 cm2). In hindsight, such abnormal values were probably caused by a persisting biofilm over plastic bars due to poor cleansing and the presence of high temperatures and humidity under indoor conditions. After the adoption of an effective cleaning protocol, a clear improvement of clinical conditions and better innate immunity parameters were evidenced 8 months later in pigs of the same age groups (see Table 3), albeit with significant time-related fluctuations (P < 0.001).
Problem herds: trial 2
Results are shown in Table 4. Haptoglobin values were very high at the onset of the observed syndrome while the other values remained in the normal range. Normal values of haptoglobin were deteted 15 days later, whereas serum bactericidal activity decreased between 1st and 2nd sampling and approached its normal range on day 30 (P < 0.001 for all differences). On the contrary, lysozyme and total haemolytic complement were always in the established range of normal values.
Problem herds: trial 3
Serological, molecular and virological tests did not confirm a possible role of specific pathogens (PRRSV, PRV, SIV, PCV2) under the observed disease conditions. Therefore, four sows of the same farrowing room and their litters were selected on the basis of low BCS values (< 2) (English et al., 1982) and blood samples were collected. Clinical immunology tests showed profound alterations of the parameters under study (Table 5). On the basis of these results, specific measures were adopted to provide satisfactory piglet comfort: wooden foils were set on the floor under heating lamps to keep temperature levels suited for piglets (around 30°C); the heating system in the farrowing rooms was improved to keep a narrow range of temperature fluctuations (18° – 20°C); the floor under the farrowing crates was no longer rinsed in order to reduce humidity. Two months later, the same parameters were investigated once again (Table 5); much better levels were observed in line with the overall clinical data.
Problem herds: trial 4
Results are shown in Table 6 as a comparison between the “problem” and the healthy herd under study. Animals of the “problem” herd (100-day old on average) showed a significantly higher serum haptoglobin concentration (P<0.01) two weeks after the onset of a respiratory syndrome sustained by Actinobacillus pleuropneumoniae. 3 months later (time 3), animals still showed significantly worse values of both SBA and serum lysozyme. High levels of serum lysozyme as well as low SBA values were measured though in both herds under study at different intervals. These results demanded to investigate further biochemical parameters, which could characterize the two herds under study. Reduced serum levels of iron and zinc were evidenced in the “problem” herd, the greatest differences being revealed at time 3 (p<0.001); animals of the “problem” herd also showed lower albumin concentrations at times 1, 2 and 3 (p< 0.001 at time 2) (data not shown).

DISCUSSION

For clinical purposes, there is a case for accurate and cheap tests of innate immunity functions in pigs, endued with descriptive and predictive value about the health and welfare status of the herd. In turn, at the lab level, procedures should prove suited for large-scale testing and the relevant assays should meet fundamental demands of sensitivity, specificity, as well as of robustness and user-friendly layout. In particular, it was our understanding to adopt laboratory parameters unlikely to be substantially affected by pig breed differences and acute, transient stressors; also, they had to be valuable as readout of chronic stress conditions. These tenets were actually confirmed by our findings in both healthy and “problem” herds. Most important, the clinical immunology tests under study were shown to vary under different environmental conditions.
The reference values assessed in healthy pigs were actually a compromise between the diverging needs of accuracy and practicality of use by swine practitioners, in that they summarized findings obtained in different age groups. Yet, they proved useful in defining the process of poor environmental adaptation and disease onset, as well as the introduction of better housing conditions. With regard to the other parameters under study, The reference values of lysozyme reflected the low serum levels generally found in healthy piglets up to 6 weeks of age; the low reference SBA value (40%) was actually dictated by the rapid decrease of its physiological values in the prodromic phases of multi-factor diseases in weaners (Table 2).
On the whole, abnormalities of the innate immune parameters were clearly related to distinct features of chronic stress in pigs. Among the immunological data under study, SBA (trial 1) was low before disease onset in connection with poor housing conditions; SBA was also low after disease onset in trial 2, along with the establishment of physiological haptoglobin values. In trial 3, all the clinical immunology parameters under study showed abnormalities under conditions of chronic thermal stress; much better values were detected once the stressor was removed, even though a caveat should be expressed about the limited number of examined samples. Interestingly, serum lysozyme was most affected by thermal stress and its values showed the largest changes in both sows and piglets when the stressing condition was on the wane ( Table 5). In trial 4, serum haptoglobin concentration was higher in fattening pigs during a respiratory syndrome sustained by A. pleuropneumoniae and a trend to moderately high serum levels was evidenced in later samplings, as well. Animals showed signs of a negative acute phase response (serum albumin concentrations) (Murata et al., 2004) and of a steady-state inflammatory condition in terms of reduced iron and zinc levels (Tayek and Blackburn, 1984). Interestingly, animals of both “problem” and healthy herd in trial 4 showed low SBA and high lysozyme concentrations in later samplings (Table 6), as a possible outcome of difficult adaptation during the fattening phase.
Our study has not formally proved a cause/effect relationship between the environmental conditions observed in the trials and the parameters under study. In fact, the format of the study (random samplings of animals at different time intervals) does not allow for such a conclusion. Our results rather showed the levels of some innate immunity parameters measured in different environments, the suitability of such parameters in characterizing poor animal health and welfare, as well as the changes observed after introduction of better environmental conditions. Owing to the above, swine practitioners and Officers of the National Veterinary Services could monitor animal health and welfare in pigs by combining clinical and environmental inspections with proper investigations of innate immunity in both breeding and fattening pigs. As for the extent of samplings, pig groups at risk should be defined on the basis of both anamnestic data and environmental inspections. The sampling strategy should give in our opinion a 95% probability of detecting alterations of clinical immunology parameters, if these are present in 20% or more of the subjects. This implies the random collection of e.g. 14 blood samples in groups of 200-1000 pigs, in the presence of a theoretical 100% sensitivity of the adopted assays (Dohoo et al., 2003). Risk assessment officers could also assist in determining further appropriate sampling levels considering a lower estimated prevalence.
The monitoring scheme described in our study demands detailed inspections in the field, a proper reporting system and follow-up phases to be run in strict co-operation between swine practitioners and farm owners. Yet, the greater insight into environmental adaptation and the greater potential for disease control in the herd can justify the adoption of such a strategy on a sound cost/benefit analysis. Most importantly, such an intervention may be conducive to a new, enticing role of the veterinary profession, in line with fundamental expectations of legislators, stakeholders and consumers about food safety.

ACKNOWLEDGEMENTS

The authors wish to thank Drs. P. Candotti and M. Tranquillo (IZSLER, Brescia, Italy) for the excellent co-operation in blood samplings from pigs of healthy herds (WEB data base) and in the relevant statistical analyses, respectively. The skilful technical assistance of Mr. G. Salvetti is gratefully acknowledged. This study was partly funded by a EU EPIZONE grant.

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TABLES

Table 1. Assessment of normal values on healthy pigs of a farrow-to-finish herd: groups under study

SOWS

FATTENING PIGS

Moving from gestation to farrowing stalls 

Weaning  (moving from farrowing to nursery stalls)

Milk production period  

Mixing  (moving from nursery stalls to growing-finishing stalls)

Moving from farrowing to mating stalls

Finishing (formation of new groups)

 

Table 2. Trial 1: time-course of clinical immunology parameters in pigs, phase one

 

Pigs  1  day

Pigs 15  days

Pigs 30–40 days

Pigs 50–60 days

Pigs 80–90 days

P value

SBA (%)

59.35  ±  12.3

15.85  ±  6.27

21.81  ±  9.56

18.46  ±  8.72

20.24  ±  9.03

< 0.001

Complement (C’H50/ 0.1 ml)

52.45  ±  8.31

52.53  ±  7.26

44.57  ±  10.04

48.15  ±  8.46

23.46  ±  9.67

< 0.001

Lysozyme

(μg/ml)

2.8   ± 1.02

2.67 ±  1.12

3.72 ±  1.14

3.12 ±  1.5

5.63 ± 1.61

< 0.001

20 blood samples were randomly collected from the same group of pigs in each of the reported age groups. Results are shown as mean values ± 1 standard deviation. ANOVA and Newman-Keuls Multiple Comparison Test: P < 0.001 for SBA (1 vs. 15 day-old piglets), complement and lysozyme (50/60 vs. 80/90 day-old piglets).

 

Table 3. Trial 1: time-course of clinical immunology parameters in pigs, phase two

 

Pigs  1  day

Pigs 15  days

Pigs 30–40 days

Pigs 50–60 days

Pigs 80–90 days

P value

SBA (%)

66.23 ± 12.94

56.36 ± 15.39

36.54 ± 10.31

41.24  ±  11.48

39.46  ±  15.16

< 0.001

Complement (C’H50/ 0.1 ml)

57.93 ± 5.26

54.21 ± 8.05

46.13 ±  8.29

48.15 ±  9.89

35.84 ±  9.30

< 0.001

Lysozyme (μg/ml)

2.14± 0.91

1.64 ±0.65

3.56 ±  1.7

2.21 ±  1.31

3.83 ±  1.83

< 0.001

8 months after the survey reported in Table 2, twenty blood samples were randomly collected in the same herd from pigs in each of the reported age groups. Results are shown as mean values ± 1 standard deviation. ANOVA and Newman-Keuls Multiple Comparison Test: P < 0.001 for SBA (30/40 vs. both 1 and 15 day-old piglets), complement (80/90 vs. both 50/60 and 30/40 day-old piglets) and lysozyme (30/40 vs. 15 day-old , 80/90 vs. 50/60 day-old piglets).

 

Table 4. Trial 2: A. pleuropneumoniae infection and innate immune response

Parameters  under study

1st  sampling Disease onset

2nd  sampling - 15 days later

3rd sampling - 30 days later

P value

Haptoglobin

(mg/ml)

4.92 ± 1.41

0.76 ± 0.7

0.59 ± 0.52

< 0.001

Serum Lysozyme

(μg/ml)

1.67 ± 0.56

1.86 ± 0.49

1.92 ± 0.45

> 0.05

Total Haemolytic Complement 

(C’H50/ 0.1 ml)

62.50 ± 8.84

55.81 ± 9.56

57.25 ± 9.16

> 0.05

SBA

 ( %)

46.29 ±15.98

9.16 ± 5.58

24.5 ± 9.5

< 0.001

20 serum samples were collected at the indicated intervals from a group of pigs (growers, > 40 Kg body weight), affected at 140 days of age (on average) by acute pleuro-pneumonia sustained by A. pleuropneumoniae. Results are reported as mean values ± 1 standard deviation. ANOVA and Newman-Keuls Multiple Comparison Test: P < 0.001 for haptoblobin (1st vs. 2nd and 3rd sampling) and SBA (all differences).

 

Table 5. Trial 3: clinical immunology parameters in pigs exposed to thermal stress

A) At the beginning of the trial (time 1)

  PIGS

 Serum Bactericidal Activity (%)

 Haemolytic complement   Activity (C’H50/0.1 ml)

Serum  Lysozyme (mg/ml)

Sows (1st – 5th farrowing)   

37.6 ± 6.2

38.8 ± 25.1

3.6 ± 1.2

Piglets (10 – 13 days of age)

16.0 ± 6.9

36.0 ± 7.4

4.7 ± 0.7


B) Two months later, under satisfactory thermal conditions (time 2)

PIGS

Serum bactericidal activity (%)

Haemolytic complement activity

(C’H50 / 0.1 ml)

Serum lysozyme

(mg/ml)

Sows (2nd – 6th farrowing)

45.8 ± 4.6

46.9 ± 9.4

1.8 ± 0.3

Piglets (16 – 18 days of age)

29.7 ± 7.7

51.7 ± 2.7

2.2 ± 0.6

Clinical immunology parameters were investigated in four sows and as many piglets (one per litter) at the beginning of the trial (panel A) and 2 months later after major improvements of the housing conditions (panel B). Results are expressed as mean ± 1 standard deviation. Two-way ANOVA: both animal category (sows and piglets) and treatment (time 1 vs. time 2) effects were included in the analysis; the differences between time 1 and 2 are significant for both SBA and lysozyme (P < 0.01). The differences between sows and piglets are significant for SBA, only (P < 0.01).

 

Table 6. Trial 4: time-course of clinical immunology parameters in fattening pigs

 

Time 0,

 healthy herd

Time 0,

 “problem” herd

Time 1,

 healthy herd

Time 1,

 “problem” herd

 SBA %

40.49 ± 31.98a

27.10 ± 14.28 a

25.85 ± 14.89 a

36.31 ± 27.59 a

 C’H50 / 0.1 ml

37.05 ± 26.30 a

53.82 ± 14.84 a

50.05 ± 15.0 a

55.59 ± 11.47 a

Lysozyme µg/ml

5.22 ± 1.72 a

5.65 ± 2.10 a

7.22 ± 2.34 a

8.70 ± 5.31 a

Haptoglobin mg/ml

0.70 ± 0.27 a

1.71 ± 1.23 b

0.89 ± 0.33 a

0.77 ± 0.21 a

 

Time 2,  healthy herd

Time 2, “problem” herd

Time 3,

 healthy herd

Time 3,

 “problem” herd

SBA %

25.20 ± 10.85 a

37.20 ± 24.86 a

26.54 ± 11.86 a

13.86 ± 11.74 b

 C’H50 / 0.1 ml

62.38 ± 0.26 a

61.29 ± 2.40 a

47.12 ± 11.22 a

56.38 ± 11.61 a

Lysozyme µg/ml

6.15 ± 1.43 a

3.95 ± 1.64b

2.01 ± 0.45 a

5.70 ± 2.59 b

Haptoglobin mg/ml

1.32 ± 0.75 a

2.34 ± 1.91 a

1.93 ± 2.04 a

2.57± 1.9 a

The time-course of clinical immunology parameters was investigated in age-matched, growing-finishing pigs of a healthy and a “problem” herd, respectively. Animals of the “problem” herd were affected by a respiratory syndrome sustained by A. pleuropneumoniae starting two weeks before time 0. Blood samplings were carried out on 15 pigs per herd (random choice) at 30-day intervals at times 0, 1, 2 and 3, respectively. Results are expressed as mean ± 1 standard deviation. By 2-way ANOVA significant differences among time points (P < 0.05) were demonstrated for the 4 parameters under study. Herd was shown to exert a significant effect on complement and haptoglobin values (P < 0.01). Within a row, different superscript letters indicate significant differences at P < 0.05 for the corresponding time point, as shown by a t test for unpaired values.


 

 


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