Avian leukaemia virus (ALV), Newcastle disease virus and avian infectious Bursal disease virus cause poultry diseases and have a great impact on poultry breeding
(Shebannavar et al., 2009; Pandey et al., 2020). The avian leukosis virus (ALV-J) is an oncogenous alpharetrovirus of the Retroviridae family. Avian leukosis can cause growth retardation, immune suppression and decreases in the fertilisation rate of breeding roosters and egg laying by hens
(Cui et al., 2006). Commonly transmitted to vertical embryos through the birth canal of infected hens, ALV-J causes lifelong toxicity to infected chickens
(Li et al., 2017), thus seriously affecting and restricting the development of the poultry breeding industry
(Stedman et al.1999). ALV-J mainly induces myelocytomatosis and lymphocytic tumor, especially in the liver, kidney and bursa of Fabricius
(Payne et al., 2012; Stedman et al., 1999). It also leads to immunosuppression, causing the continuous enlargement of the spleen, thymus gland and bursa of Fabricius
(Dong et al., 2015). Moreover, the body weights of infected chicks and related organs, such as the heart and liver, are considerably altered compared with those of normal chicks and are far lower than those of normal chicks, indicating that the virus inhibits growth.
In the entire duration of the challenge, the infected chicks showed signs of cowardice and listlessness. Vivisection revealed a marked dark enlargement of the caecum terminal of 42-day-old chicks and thymus tissues abnormally increased in size, but no obvious morphological abnormality was observed. Meanwhile, the bursa of Fabricius, an immune organ unique to birds, was many times larger than normal. The mass concentration of cytokines in the challenge group showed a trend of increasing initially and then decreasing. The peaks of IL-2 and IFN-g were detected at 21 d.p.i and the peak of IL-4 was detected at 28 d.p.i
(Zhu et al., 2017). The expression levels of related cytokines, such as IL-6, IL-18, IFN-a and IFN-g, in different immune organs, such as the spleen and thymus showed a rapid increase in the early stage of the challenge and a sharp decline after reaching their respective peak values. However, the expression pattern of the bursa of Fabricius is quite different from that of the spleen
(Gao et al., 2015). The enhancement of these diseases by concomitant ALV infection is most likely the consequence of immunosuppression, but the mechanism of ALV-induced immunosuppression has not been completely characterized.
This experiment examined ALV-J immunosuppression and growth inhibition in chickling as the breakthrough point and the specific effects of the artificial inoculation of ALV-J virus were investigated using a macro performance model. Moreover, differences in body weight, organ weight and immune index between in clear infection chicks and health groups were determined. ALV-J infection was explored on the basis of the immune suppression mechanism of the interaction effect of the virus and a theoretical basis was provided.
The experiment animals are specific pathogen-free White Leghorn chickens (SPF), which were purchased from Beijing Boehringer lngelheim Vita Biotechnology Co, Ltd. They were placed in an incubator and then transferred to an isolated feeder. They were transferred again to a negative pressure isolation feeder after incubation. One week before they were subjected to the experiment, they were rinsed with water and fumigated with formaldehyde. A clean feeding environment and sufficient ventilation were ensured. All the chickens were free to eat and drink. The feed of the SPF chickens was sterilised with ultraviolet ray and a small amount of multivitamin was added to drinking water. The 1–14-day-old chickens were fed eight times and given water two times a day. Body temperature was recorded nine times a day. Meanwhile, the 15–42 day-old chickens were fed three times (8:00 am, 2:00 pm and 9:00 pm) and given water two times. Body temperature was recorded three times. The whole breeding and research experiment process of the chickens was carried out in the College of Animal Science and Technology, Southwest University from March to August, 2019.
ALV-J strain (NX0101) used in the experiment was provided by ShanDong Agricultural University (genbank accession nmber: DQ115805.1). One-day-old chicks in the experimental group were infected with the ALV-J virus through the intraperitoneal injection of 0.2ml (about 103TCID50) of virus solution
(Dong et al., 2012). DF-1 cells were used for ALV-J virus culture in an environment of 37oC and 5% CO2. The control group was subjected to simulated infection and each chick was intraperitoneally injected with 0.2 ml of DMEM culture solution. Tissues were collected for PCR amplification detection.
At 3, 5, 7,14, 28 and 42 days, five chicks from the experimental group and another five from the control group were randomly selected for weighing. After the chicks were euthanised, bursa of fabricius, thymus, liver, heart and spleen were collected for weighing. Liver RNA was extracted and retranscribed to cDNA with a Takara PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time). The composition and conditions of RNA reverse are given in Table 1 and 2 respectively. PCR amplification was performed using cDNA as template. The primers H5 and H7 reported in the literature were used for PCR amplification
(Smith, et al., 1998). The contents and conditions of PCR are given in Table 3 and 4 and 5 respectively. The PCR product was sequenced and the sequencing data were analysed through blast analysis (Blast results are attached in Attachment 1).
The immune organ index was calculated using the formula immune organ index = immune organ weight (g) / chicken live weight (g) and the corrected index was calculated in the normal group. The formula was correcting immune index = immune organ weight (g) of the challenge group / organ weight (g) of the control. The significance of the variability among the trials was analysed with SPSS24.0. The results were presented as means ± SEM and statistical significance was represented by P values of < 0.05, 0.01.
Morphological analysis
The healthy chicks in the control group showed good growth and development, soft feathers and excellent mental state. By contrast, the chicks in the experimental group were stunted, had messy and fried feathers, were unstable or unable to stand or limp and showed depressed spirit and abnormal uplift of the sternum. The growth and development status of each chick in the experimental group varied. The liver and spleen were swollen, the bursa of Fabricius was swollen and the cecum was swollen and black. As shown in Fig 1, the organs of the chickens in the test group had cysts and the number of infections in the chickens increased over time.
Virus detection by PCR
Liver tissues were used for the extraction of total RNA, which was then reverse-transcribed into cDNA for PCR amplification. The electrophoresis results showed that the amplification band was single and a 545 bp gene fragment was obtained from the sample, which was consistent with the expected fragment. The sequenced PCR products were consistent with the target fragment according to the blast analysis results. All the chicks in the test group successfully challenged the virus, as shown in Fig 2.
Weight curves of immune organs
As shown in Fig 3 A and C, at the early stage of the challenge, no significant differences in the weight of thymus and bursa of Fabricius were observed between the experimental and control groups and the change curve was nearly the same. The weights of the thymus and bursa of Fabricius increased rapidly compared with those in the control group, whereas those in the experimental group increased slowly. After 21-28 days, the weights of the thymus and bursa of Fabricius of the chicks in the experimental group began to increase rapidly and reached their peak values at 28 days but were consistently lower than those of the control group. Between 28 and 42 days, the weights of the thymus and bursa of Fabricius of the experimental group began to decrease rapidly and significantly, whereas those of the control group showed a steady increase. As shown in Fig 3B. The appearance of the spleen was different from the appearances of the bursa of Fabricius and thymus. The spleen quality of the challenge group increased faster than that of the control group from the early stage of infection to 21 days and was higher than that of the control group. After 21 days of challenge, spleen mass increased sharply and was more than 2.5 times that of the control group and then decreased sharply at 28 days until it was the same as that of the control group in the 33th day. A low level of inhibition was observed.
Weights of other organs (liver and heart) and body weight curve
As shown in Fig 4, the weights of the liver, heart and body of the chicks in the control group were all higher than those in the experimental group at each period. Within 14 days of challenge, weight difference was non-significant and the liver and heart were in the slow growth period. The relevant organs and body weight of the experimental group and the control group were in the stage of rapid growth, but the liver, heart and body weight of the control group significantly changed, showing that the weights in the control group were much higher than those in the experimental group. The liver, heart and body weight of the chicks in the experimental group reached their maximum values at 28 days and body weight was basically unchanged. The weights of the heart and liver continued to decline, showing a state of gradual atrophy.
Effects of ALV-J on thymus, bursa of Fabricius and spleen index of chicks
The spleen, bursa of Fabricius and thymus were considered the most important immune organs involved in the body’s humoral and cellular immunity. The sizes of immune organs were usually used in evaluating the immune status of chickens
(Rivas et al.1988).
Fig 5 show the difference among the immune organ indexes of the challenge and control groups after days of ALV infection (21, 28 and 42 days). From day 21 to day 42, the immune organ indexes (thymus, spleen and bursa of fabricius) of the challenge and control groups showed significant differences (P<0.05), but the spleen index showed no difference at day 42.
Comparison of correcting immune index
As shown in Fig 6, after 14 days of challenge, the body weight of the experimental group was significantly different from that of the control group, showing growth inhibition. In the experimental group, the immune-correction index of the spleen was always greater than 1 after challenge and reached a peak of 2.8 at 21 days. The main manifestation was the continuous enlargement of spleen, which was alleviated over time. The immunological correction number of the thymus or bursa of Fabricius was basically less than 1 (except that the thymus was greater than 1 in the initial time of challenge) from the challenge treatment to 42 days after treatment and the thymus was basically in the state of organ atrophy and immunosuppression compared with that in the control group. The liver and heart did not differ significantly from the control group, but the heart showed slight atrophy. The weight of the spleen varied significantly, whereas the weights of the bursa of Fabricius and thymus showed little changes.
Fig 3 B and 5 show that the change trend of spleen quality in the challenge group is slow increase in the early stage, steep increase in the middle stage and finally a sharp decline. In addition, the spleen quality of the challenge group was always higher than that of the control group 33 days after the challenge. After this period, the mass ratio was reversed. Interestingly, the pattern of change was similar to patterns of related cytokines in the spleen
(Gao et al., 2015). These findings indicate that the spleen plays a momentous role in the interaction between ALV-J and the immune system. We also observed that the quality of the thymus and bursa of Fabricius in the challenge group was lower than that in the control group during the whole test period but showed a sharp rise and then a sharp decline. Such changes in quality were consistent with those in cytokine levels in corresponding tissues
(Gao et al., 2015).
We hypothesized that the patterns of differences in the spleen, thymus and bursa of Fabricius might be due to differences in organic structure and function. As the largest lymphoid organ in the body, the spleen not only produces a large number of lymphocyte macrophages and monocytes but also produces immunoglobulin, complement and other immune substances. Therefore, humoral immunity mediated by the early stage of spleen and the cell-mediated immunity mediated by the middle stage play important roles in resistance against and elimination of ALV. The liver and heart, as one of the largest internal organs, play important roles in growth and metabolism. Our data revealed that the weights of the heart and liver in the infected group were significantly lower than those in the control group, showing significant differences.
