Heat stress, Eimeria spp. and C. perfringens infections alone or in combination modify gut Th1/Th2 cytokine balance and avian necrotic enteritis pathogenesis.

Information on the dynamics of the chicken immune system during bacterial or parasite challenge in the presence or absence of stressful situations may provide a better understanding of the complex mechanisms behind these diseases. Necrotic enteritis (NE) had been controlled previously by the proper use of antimicrobial agents; however, more recently, NE has reemerged in many countries. The imposed restrictions on antimicrobial use and/or the intensive productive programs implemented by producers are challenges the birds, leading to large host adaptive responses that in many instances are like those elicited by stressors. This study analyses the effects of heat stress on Th1/Th2 cytokine balance, pathological features, and Toll-like receptor expression in the small intestine of broiler chickens infected with Clostridium perfringens type A in the presence or absence of Eimeria spp. co-infection. This co-infection model was experimentally used because it reproduces the findings commonly observed in the field during avian NE. For this purpose, broiler chickens infected with C. perfringens and/or Eimeria spp. were reared in isolator chambers subjected or not to heat stress intermittently. It was observed that heat stress directs the expression of Th2-type cytokines, increases Toll-like receptor 4 expression in the intestine and reduces the disease severity induced by Eimeria spp. and C. perfringens infections alone or in combination, most likely as a consequence of stress-induced changes in brain-gut axis activity.

Heat stress reduces Eimeria spp. infection and interferes with C. perfringens infection via activation of the hypothalamic-pituitary-adrenal axis.

Heat stress has a relevant effect on animal health and productivity. Stress and environmental changes can contribute to disease development, such as avian necrotic enteritis (NE). The goal of this study was to analyze the effects of heat stress applied to broiler chickens in an experimental model of co-infection with Clostridium perfringens and Eimeria spp. Therefore, the current study was designed to analyze the effect of heat stress to broiler chickens in an experimental model of infection or co-infection with Clostridium perfringens and Eimeria spp. C. perfringens was given in the poultry feed and the Eimeria infection was induced by gavage with a live oocysts vaccine dose 30 times higher than the manufacturer recommendation. We observed a reduction in the secretory IgA concentration in the jejunum and ileum in heat-stressed chickens compared to non-stressed chickens. Decreased maximum scores of intestinal necrosis, crypt abscesses and transmural lesions were observed in the heat-stressed chickens co-infected and infected with Eimeria compared to the respective unstressed groups. Heat stress caused an increase the intestinal lesion scores in chickens infected with C. perfringens only. The crypt depth was greater in chickens from the heat-stressed groups compared to the non-stressed groups. We also demonstrated that HS decreased infection and/or Eimeria development in the intestinal epithelium, reducing the harmful potential of C. perfringens.

Heat Stress Modulates Brain Monoamines and Their Metabolites Production in Broiler Chickens Co-Infected with Clostridium perfringens Type A and Eimeria spp.

Heat stress has been related to the impairment of behavioral and immunological parameters in broiler chickens. However, the literature is not clear on the involvement of neuroimmune interactions in a heat stress situation associated with bacterial and parasitic infections. The present study evaluated the production of monoamines and their metabolites in brain regions (rostral pallium, hypothalamus, brain stem, and midbrain) in broiler chickens submitted to chronic heat stress and/or infection and co-infection with Eimeria spp. and Clostridium perfringens type A. The heat stress and avian necrotic enteritis (NE) modulated the neurochemical profile of monoamines in different areas of the central nervous system, in particular, those related to the activity of the hypothalamus-hypophysis-adrenal (HPA) axis that is responsible for sickness behavior. C. perfringens and/or Eimeria infection, heat stress increased 5-hydroxytryptamine (5-HT), 4,4 dihydroxyphenylacetic acid (DOPAC), and DOPAC/dopamine (DA) in the rostral pallium; 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), homovanillic acid (HVA), HVA/DA, DOPAC/DA, and 5-hydroxyindoleacetic acid (5-HIAA)/5-HT in the hypothalamus; MHPG, 5-HIAA/5-HT, DOPAC/DA, and HVA/DA in the midbrain; and MHPG, DOPAC, HVA, HVA/DA, DOPAC/DA, and 5-HIAA/5-HT in the brainstem. Heat stress decreased noradrenaline + norepinephrine (NOR + AD) in all brain regions analyzed; 5-HT in the hypothalamus, midbrain, and brainstem; and DA in the midbrain. The results also showed the existence and activity of the brain-gut axis in broiler chickens. The brain neurochemical profile and corticosterone production are consistent with those observed in chronic stressed mammals, in animals with sickness behavior, and an overloading of the HPA axis.

The gut-brain axis interactions during heat stress and avian necrotic enteritis.

The gut-brain axis is known to modulate behavioral and immune responses in animals; evidence supporting this modulation in chickens, however, is elusive. Here, we analyzed the effects of heat stress and/orClostridium perfringens (CP) infection on behavior, intestinal morphology, brain activity, and corticosterone serum levels in chickens. Broilers were randomly divided into 5 equal groups: a naïve group (N), a thioglycolate group (T), a thioglycolate heat-stressed group (T/HS35), an infected group (I), and an infected/stressed (I/HS35) group. Broilers in the I and I/HS35 groups were experimentally infected withClostridium perfringensfrom the 15th to the 19th day of life. Heat stress (35±1°C) was constantly applied to the broilers in the stressed groups from the 14th to the 19th day of life. Our data showed that heat stress andC. perfringensinfection produced significant differential responses in the chickens' behavior and in c-fosexpression in the paraventricular nucleus of the hypothalamus (PVN), nucleus taenia of the amygdala (Tn), medial preoptic area (POM), andglobus pallidus (GP) of the chickens. Heat stress ameliorated some of the intestinal lesions and the neuroendocrine changes induced byC. perfringensin the birds. Our results suggest the existence of clear relationships between the degree of intestinal lesions, the chickens' behavioral outcomes, brain activity, and serum levels of corticosterone. Together, they reinforce the importance of neuroimmunomodulation and especially of brain-gut axis interactions.

Effects of heat stress on the formation of splenic germinal centres and immunoglobulins in broilers infected by Clostridium perfringens type A.

Avian necrotic enteritis (NE) induced by Clostridium perfringens is a disease that affects mainly the first weeks of poultry's life. The pathogenesis of NE is complex and involves the combination of several factors, such as co-infection with different species of coccidia, immunosuppression and stress. Stress is one of the main limiting factors in poultry production. Although several studies emphasized the effects of stress on immunity, few works analyzed these effects on immunoglobulins and on germinal centres (GCs), which are specialized microenvironments, responsible for generating immune cells with high affinity antibodies and memory B-lymphocytes. Thus, the effects of heat stress associated or not with thioglycolate broth culture medium intake and/or C. perfringens infection on corticosterone serum levels, spleen GCs development and immunoglobulin production in broilers were evaluated. Results showed that heat stress, thioglycolate and C. perfringens per se increased corticosterone serum levels, although this was not observed in heat stressed and thioglycolate and C. perfringens-treated chickens. The serum levels of IgA, IgM and IgY were differently affected by heat stress and/or infection/thioglycolate. Heat stress decreased the duodenal concentrations of sIgA, which was accompanied by a reduction in GCs number in the duodenal lamina propria; a trend to similar findings of sIgA concentrations was observed in the chickens' jejunum. Changes in spleen and Bursa of Fabricius relative weights as well as in spleen morphometry were also noted in heat stressed animals, infected or not. Together, these data suggest that heat stress change GCs formation in chickens infected or not, which that may lead to failures in vaccination protocols as well as in the poultries' host resistance to infectious diseases during periods of exposure to heat stress.

Effects of heat stress on peripheral T and B lymphocyte profiles and IgG and IgM serum levels in broiler chickens vaccinated for Newcastle disease virus.

Multiple factors, such as environment, nutritional status, and disease, induce stress in animals during livestock production. It has been shown that poultry exposed to stressors for prolonged periods had decreases in their performance parameters, mortality and decreased host resistance to pathogenic agents. It seems that early age stress may have long-lasting impact and could possibly modify the expression of their genetic potential on growth performance and immunity. This study aimed to discuss the effects of early-age heat stress on the blood lymphocyte phenotypes (B and T lymphocytes) and plasma immunoglobulin levels (IgM and IgG) in chickens vaccinated against paramixovirus of the Newcastle (NC) disease (LaSota strain). For this purpose, 96 male chickens (Cobb) were divided into 4 groups: 1) control (C), 2) heat-stressed (HS), 3) control vaccinated (C/V), and 4) heat-stressed and Vaccinated (HS/V). The NC vaccine was administered twice on experimental day (ED) 7 and ED14, and the heat stress (38 ± 1°C) was applied from ED2 to ED6. The data showed that HS increased the corticosterone serum levels in the HS group compared with the control groups (C and C/V groups). At ED7, increased concentrations of IgM were observed in birds in the HS and HS/V groups compared with C and C/V animals; chickens from the HS/V group presented increased IgG levels compared with those in the birds of the C group. The heat stress shifted the immune cell profile from B-lymphocyte to a T-cytotoxic and T-helper lymphocyte profile, and this immune cell pattern persisted until the end of the study period. It was concluded that heat stress immunomodulated the immune function response of the chickens to the NC disease vaccine challenge.

Effects of cold stress and Salmonella Heidelberg infection on bacterial load and immunity of chickens.

We analysed the effects of cold stress (19 ± 1°C, 6 h /day, from the first to the seventh day of life) applied to specific pathogen free (SPF) chickens. On experimental Day 1 (ED1), chicks were divided into four groups: C (not infected and kept under thermoneutral condition); CS (not infected and cold stressed); PC (Salmonella Heidelberg (SH) infected and kept under thermoneutral condition) and PCS (SH infected and cold stressed). High concentrations of corticosterone were found in the cold stressed birds on ED7 and ED21, with a greater increase in birds of the PCS group. Stress or non-stressed SH-infected birds had high levels of norepinephrine on ED21. On ED21, an increased percentage and number of SH were found in birds of the PCS group. On ED7, a decrease in macrophages presenting MHCII, CD8(+) and CD8(+) γδ cells was observed in the chickens of the CS group. Decrease was observed in CD3(+) cells in the birds of the PCS group and increase in macrophages presenting MHCII cells and of the CD4(+)/CD8(+) ratio in chickens of the CS group on ED21. There was a decrease in CD8(+) γδ cells in birds of the CS group on ED21 and in the CD3(+) and CD8(+)cell numbers in chickens of the PCS group on ED21. Our results suggest that cold stress applied to chickens in the first 7 days of life increases both the hypothalamus pituitary adrenal axis and the sympathetic nervous system activities, leading to long-term immune cell dysfunction, thus allowing increased SH invasion and persistence within the birds' body.

Effects of long-term heat stress in an experimental model of avian necrotic enteritis.

Stressful conditions are predisposing factors for disease development. Heat stress is one of the most important stressors in poultry production. The reemergence of some previously controlled diseases [e.g., avian necrotic enteritis (NE)] has been extensively reported. The combination of bacterial infection and certain environmental factors have been reported to trigger the disease. The aim of this study was to analyze the effects of long-term heat stress (35 ± 1°C) on the development of NE in broiler chickens. For this purpose, 60 male broiler chickens were divided into the following 6 groups: control group (C), heat stressed control group (C/HS35), thioglycolate group (T), thioglycolate heat-stressed group (T/HS35), infected group (I), and infected heat-stressed group (I/HS35). The poultry of groups I and I/HS35 were experimentally infected with Clostridium perfringens via their feed from 15 to 21 d of life. Heat stress (35 ± 1°C) was constantly applied to the birds of the stressed groups from 14 to 21 d of life. The infected and heat-stressed broiler chickens presented a trend toward a decrease in gross lesion scores and significantly lower microscopic scores of necrosis in the duodenum and jejunum (P < 0.05), lower fusion of villi in the duodenum (P < 0.05), and lower congestion scores in the jejunum and ileum (P < 0.05) in relation to infected and non-heat-stressed chickens. Broilers of I/HS35 group also exhibited small number of heterophils in the duodenum and jejunum compared with those of the I group (P < 0.05). Furthermore, the duodenum and jejunum of infected and heat-stressed broilers showed lower number of clostridia on the intestinal mucosa (P < 0.05). Data were discussed in light of a heat stress induced reduction on intestinal inflammation via a decrease in heterophil migration to the intestinal mucosa, which in turn might have reduced tissue damage during inflammation, hence preventing the development of a more severe form of NE.