The objective of this study was to investigate the kinetics of Hsp60, Hsp70, Hsp90 protein, and messenger RNA (mRNA) expression levels and to correlate these heat shock protein (Hsp) levels with tissue damage resulting from exposure to high temperatures for varying amounts of time. One hundred broilers were heat-stressed for 0, 2, 3, 5, and 10 h, respectively, by rapidly increasing the ambient temperature from 22 ± 1°C to 37 ± 1°C. Obvious elevations of plasma creatine kinase indicate damage to myocardial cells after heat stress. Hsp70 and Hsp90, and their corresponding mRNAs in the heart tissue of heat-stressed broilers, elevated significantly after 2 h of heat exposure and decreased quickly with continued heat stress. However, the levels of hsp60 mRNA in the heart of heat-stressed broilers increased sharply (P < 0.01) at 2 h of heat stress but then decreased quickly after 3 h, while the level of Hsp60 protein in the heart increased (P < 0.01) at 2 h of heat stress and maintained a high level throughout heat exposure. The results indicate that the elevation of the three Hsps, especially Hsp60 in heart, may be important markers at the beginning of heat stress and act as protective proteins in adverse environments. The reduction of Hsp signals in the cytoplasm of myocardial cells implies that myocardial cell lesions may have an adverse impact on the function of Hsps during heat stress. Meanwhile, the localization of Hsp70 in blood vessels of broiler hearts suggests another possible mechanism for protection of the heart after heat exposure.

1. In order to understand the distribution and variation of Hsp90 and its corresponding mRNA in the tissues of heat-stressed broilers, 100 experimental birds were heat stressed for 0, 2, 3, 5 and 10 h. 2. Both nuclear and cytoplasmic Hsp90-positive signals were observed in myocardial cells, hepatocytes and renal tubule epithelial cells. The expression of Hsp90 increased in the heart, liver and kidney of broilers after exposure to high temperature for 2 h. 3. Following non-significant reduction after 3 and 5 h in the treatment groups, the expression of Hsp90 in the heart and kidney, but not in the liver, increased again after 10 h of heat stress. In the heart and kidney, hsp90 mRNA transcription levels exhibited the same trend as the protein expression of Hsp90. 4. Induction of hsp90 mRNA and Hsp90 protein at an early stressing stage indicated that heat stress can directly stimulate and quickly initiate the transcription of hsp90 mRNA and translation of Hsp90 protein to protect cells. 5. Hsp90 was strongly expressed in endothelial cells and blood vessel walls, suggesting that there is a relationship between vasoconstriction and Hsp90 localisation, which influences blood flow regulation.

Heat stress negatively affects poultry production and animal health. In response, animals invoke a heat stress response by inducing heat shock proteins (HSPs). Scientists are actively seeking natural products that can enhance the heat shock response. The present study aimed at assessing the effects of a purified rosemary extract comprising antioxidant compounds on the heat shock response and HSP expression profile in broiler chickens. The response of broilers to HS in the presence of purified rosemary extract was assessed using an in vivo myocardial cell model. Pathological lesions of heart tissue were examined microscopically. The levels and activities of enzymes associated with heart damage and oxidative damage were detected. Immunohistochemical staining was performed for HSPs in myocardial cells. The results showed that lactate dehydrogenase (LDH), creatine kinase (CK), and myocardial CK (CKMB) levels were reduced by the purified rosemary extract before and during heat stress. Heat stress alone increased CK and CKMB levels. The levels of oxidative damage‐associated enzymes were compared between the rosemary + heat stress and heat stress‐alone groups. The results indicated that in terms of these enzymes, the purified rosemary extract induced a more antioxidative state. Pathological examinations showed that heat stress caused myocardial fiber fracture, karyopyknosis, and degeneration. The addition of purified rosemary extract ameliorated these lesions to some degree, preserving more of the basic structure. Heat stress decreased the cellular levels of crystallin alpha B (CRYAB) and HSP70. The addition of the purified rosemary extract significantly increased the levels of CRYAB and HSP70 during heat stress (p < 0.0001). Immunohistochemistry showed that after rosemary treatment, CRYAB and HSP70 showed more intense staining compared with the no heat stress control group. In the rosemary + heat group, after 10 hours of heat stress, the staining intensity of these two proteins remained higher than in the heat stress group. Thus, purified rosemary extract could induce high levels of HSP70 and CRYAB in chicken hearts before and during heat stress. Purified rosemary extract could be used to alleviate heat stress in broiler chickens.

We investigated whether acetyl salicylic acid (ASA) protects chicken myocardial cells from heat stress-mediated damage in vivo and whether the induction of Hsp27 expression is connected with this function. Pathological changes, damage-related enzyme levels, and Hsp27 expression were studied in chickens following heat stress (40 ± 1 °C for 0, 1, 2, 3, 5, 7, 10, 15, or 24 h, respectively) with or without ASA administration (1 mg/kg BW, 2 h prior). Appearance of pathological lesions such as degenerations and karyopyknosis as well as the myocardial damage-related enzyme activation indicated that heat stress causes considerable injury to the myocardial cells in vivo. Myocardial cell injury was most serious in chickens exposed to heat stress without prior ASA administration; meanwhile, ASA pretreatment acted protective function against high temperature-induced injury. Hsp27 expression was induced under all experimental conditions but was one-fold higher in the ASA-pretreated animals (0.3138 ± 0.0340 ng/mL) than in untreated animals (0.1437 ± 0.0476 ng/mL) 1 h after heat stress exposure, and such an increase was sustained over the length of the experiment. Our findings indicate that pretreatment with ASA protects chicken myocardial cells from acute heat stress in vivo with almost no obvious side effects, and this protection may involve an enhancement of Hsp27 expression. However, the detailed mechanisms underlying this effect require further investigation.

Heat stress is exacerbated by global warming and affects human and animal health, leading to heart damage caused by imbalances in reactive oxygen species (ROS) and the antioxidant system, acid-base chemistry, electrolytes and respiratory alkalosis. Vitamin C scavenges excess ROS, and sodium bicarbonate maintains acid-base and electrolyte balance, and alleviates respiratory alkalosis. Herein, we explored the ability of vitamin C alone and in combination with equimolar sodium bicarbonate (Vitamin C-Na) to stimulate endogenous antioxidants and heat shock proteins (HSPs) to relieve heat stress in H9C2 cells. Control, vitamin C (20 μg/ml vitamin C for 16 h) and vitamin C-Na (20 μg/ml vitamin C-Na for 16 h) groups were heat-stressed for 1, 3 or 5 h. Granular and vacuolar degeneration, karyopyknosis and damage to nuclei and mitochondria were clearly reduced in treatment groups, as were apoptosis, lactate dehydrogenase activity and ROS and malondialdehyde levels, while superoxide dismutase activity was increased. Additionally, CRYAB, Hsp27, Hsp60 and Hsp70 mRNA levels were upregulated at 3 h (p < 0.01), and protein levels were increased for CRYAB at 0 h (p < 0.05) and 1 h (p < 0.01), and for Hsp70 at 3 and 5 h (p < 0.01). Thus, pre-treatment with vitamin C or vitamin C-Na might protect H9C2 cells against heat damage by enhancing the antioxidant ability and upregulating CRYAB and Hsp70.

The global distribution of canine parvovirus (CPV-2) derived from a closely related carnivore parvovirus poses a considerable threat to the dog population. The virus is continuously undergoing genetic evolution, giving rise to several variants. To investigate the prevalence of Chinese CPV-2 strains in recent years, a total of 30 CPV-2 strains were collected from 2018 to 2021 and the VP2 gene was sequenced and analyzed. Two variants, new CPV-2a (297Ala, 426Asn) and CPV-2c (426Glu), were identified. In contrast to previous reports, the CPV-2c variant has gained an epidemiological advantage over the new CPV-2a variant in China. To compensate for the relatively small sample size, 683 Chinese CPV-2 strains identified between 2014 and 2019 were retrieved from the GenBank database and previous publications, and analyses of these strains further supported our findings, which should be considered since the CPV-2c variant has been frequently associated with immune failure in adult dogs. VP2 protein sequence analysis revealed several amino acid substitutions, including Ala5Gly, Pro13Ser, Phe267Tyr, Tyr324Ile, Gln370Arg, Thr440Ala, and Lys570Arg. Phylogenetic analysis of full-length VP2 gene indicated a close relationship between Chinese strains and other Asian strains, suggesting mutual transmission between Asian countries. Furthermore, intercontinental transmission is a cause for concern. Surprisingly, two feline panleukopenia virus (FPV) strains with the Ile101Thr mutation in the VP2 protein were identified in canine fecal samples; FPV has been considered incapable of infecting dogs. This study clarified the epidemic characteristics of Chinese CPV-2 strains detected between 2014 and 2019, offering a reference for epidemic control. In addition, the detection of FPV in canine samples may provide information for future studies on the evolution of carnivore parvoviruses.