1. Introduction
Over the years, the Pakistani poultry industry has emerged to be one of the most successful national industries, with an average annual growth rate of 7.5% since 2012 [
1]. Infectious disease outbreaks of various bacterial, viral and fungal origin, e.g., necrotic enteritis (NE), avian influenza (AI) and aspergillosis have posed a significant economical and health threat to this industry. NE, caused by
Clostridium (C.) perfringens, is an important bacterial disease of the gastrointestinal (GI) tract in birds that may cost up to 5–6 billion USD per annum the global poultry industry [
2]. This bacterium is a Gram-positive anaerobic bacillus, which resides as normal gut microbiota [
3].
C. perfringens is grouped into five toxinotypes (A–E) depending upon the type of toxins produced e.g., alpha, beta, epsilon, iota, and enterotoxin [
4]. Recently, another type “G” was described in birds producing both alpha and a pore-forming toxin known as NetB [
5]. However,
C. perfringens type A negative for
netB, has been isolated from NE infected birds [
6]. NE shows signs of dullness, diarrhea and poor body condition with gross post-mortem lesions, i.e., ballooned and hemorrhagic intestines and microscopic lesions, i.e., shortening of the villi with damaged epithelium in the intestines [
7]. Antimicrobials targeting Gram positive bacteria are effective against acute NE in poultry.
Recently, NE has significantly re-emerged due to a gradual shift in husbandry practices towards being more environmentally friendly, e.g., ban of the use of anti-microbial growth promoters (AGPs) by the European Commission (EC) of the European Union (EU) since 2006 and the human health-related antimicrobials in food animals by the United States Food and Drug Administration (FDA) since 2015 [
8,
9]. Hence, finding a safe alternative remains indispensable. Egg yolk antibodies (EYAs) or
IgYs are termed as protective antibodies produced in the egg yolk of immunized hens that can be used to neutralize specific pathogens of enteric origin and help to improve growth performance traits in infected birds [
10,
11]. Currently, EYAs have potential application in various immunodiagnostic and therapeutic approaches used in animals and humans [
12,
13]. Keeping in view the protective role of EYAs, the current study aims to evaluate the influence of anti-clostridial EYAs on the gross and microscopic lesions found in infected broiler birds.
4. Discussion
Necrotic enteritis (NE), caused by
C. perfringens type A, is an economically important infection in Pakistani poultry [
6]. Previously, up to 25.37% prevalence was reported, highlighting the need of a safe and antimicrobial-free alternative to control and treat this infection in poultry. Keeping this necessity in mind, we tried to evaluate the beneficial effects of anti-clostridial EYAs therapy, as passive immunization has proved to be a potential alternative to antimicrobial therapy in various poultry-enteric infections [
10]. Thus, anti-clostridial EYAs were administered in experimentally infected broiler birds via different routes and their potentially beneficial effects were studied, based on various clinical and histological parameters.
Birds in groups C and D significantly (
p ≤ 0.05) improved clinical and behavioral signs including physical alertness, fecal consistency and appetite compared to group B, from the fourth and fifth weeks of the experiment, although there was not a significant (
p ≥ 0.05) difference in the mortality pattern among all groups; birds in group B showed the highest mortality rate (30%) compared to groups C (15%) and D (20%). These changes in infected birds might be due to the toxins produced by
C. perfringens, which could have directly influenced GIT, which might have indirectly affected fecal consistency, appetite and, hence, overall physical alertness [
25,
26]. Formerly, Ref [
7]” investigated that oral administration with
C. perfringens @ 4 × 10
8 cfu (three times a day) for four consecutive days resulted in loss of body condition, reduced feed intake, diarrhea and depression in birds. Furthermore, Ref [
27] reported emaciation, brown colored diarrhea, depression and reduced apatite in broilers after oral infection with 2 mL broth culture of
C. perfringens @ 1.9 × 10
9 cfu/mL for three alternate days. Similarly, oral gavage of
C. perfringens administered in broilers resulted in ruffled feathers, movement reluctance, watery feces and depression [
28,
29]. All these paraments were important and observable indicators of birds’ health and well-being, which can be graded based on the evaluation system.
RW of the internal organs, i.e., liver, kidneys and jejunal parts of the small intestine, was significantly (
p ≤ 0.05) higher in group B compared to group A, while significantly (
p ≤ 0.05) lower in groups D and C compared to group B, but higher than group A, on the day 26 post-mortem examination. This meant the most damages in these organs occurred in group B and the birds in groups D and C showed significantly improved results but lesser than group A. The production of interleukin-1 during the inflammatory process might have resulted in anorexia and muscle wastage. The gut microflora is specifically targeted by the use of antibiotics to minimize the inflammatory process, but it is reported that EYA against specific neuropeptides help to stimulate the immune system to lower growth reduction [
30,
31].
The results of gross and microscopic studies in birds belonging to group B showed the presence of gross and histological alterations in various organs. Grossly, the liver and kidney were swollen and congested, while the intestines were gas-filled and had hemorrhagic mucosa with small areas of necrosis. The histological investigations of liver parenchyma showed the presence of mono-nuclear cells, cellular necrosis, diminished sinusoidal spaces and congestion. In renal parenchyma, the presence of condensed nuclei of tubular epithelial cells, congestion with dilated urinary spaces were observed. The presence of atrophied villi with damaged epithelium was observed in the intestine. The lumen of the intestines contained necrotic debris and submucosal congestion. These pathological alterations are probably due to the alpha (α) toxin produced by
C. perfringens, which activates the arachidonic acid cascade to stimulate immune activity by producing various chemical mediators such as leukotrienes, thromboxane, and different activation factors for platelets aggregation and shrinkage of blood vessels. These clostridial toxins cause the degradation of intestinal mucosal membranes and in the liver produce tissue damage after reaching through hepato-portal circulation [
26,
32]. The results of present study agreed well with findings of [
7], who reported that oral administration with
C. perfringens (4 × 10
8 cfu) produced gross lesions and histopathological changes in the intestines and liver of broilers. Furthermore, Ref [
33] found severe gross and microscopic alterations in the liver, kidney and intestine of broiler birds after oral infection with 1 mL of broth culture of
C. perfringens (3 × 10
10 cfu/mL) for five consecutive days. The birds in groups C and D had milder or absence of these gross and microscopic changes in various organs. In this aspect, Ref [
34] found that the
C. perfringens infected birds did not show typical gross and histological changes after oral gavage of anti-clostridial
IgYs @ 3 mL/bird. The basic mechanism of EYAs activity against enteric pathogens involves binding with specific pathogens, followed by immobilization and, ultimately, a decrease in their growth [
10]. The use of anti-clostridial
IgYs provides protective effects against experimental NE infection and the findings of studies [
14,
34,
35] suggested that
C. perfringens and other enteric pathogens can be targeted through passive immunization by using purified
IgY in birds.