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Effect of a mycotoxin adsorbent on the immune response of broilers

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Anaz Abuthahir Anaz Abuthahir
Director
October 28, 2019
The toxin binder mitigating the effect of Aflatoxin, well isn't that what it is supposed to do?
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May 21, 2020
How can I measure the adsorbents ameliorating effect through studying cell-mediated immunity after infection with E-coli or Salmonella in chicks feeding aflatoxins or any combination of another mycotoxin with other bacterial infections?
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May 21, 2020
Yes, I would like to know more about the effect of mycotoxin adsorbents on the immune response of broilers.
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Manuel Contreras Manuel Contreras
Doctor of Veterinary Medicine
Special Nutrients Special Nutrients
Florida, United States
May 22, 2020
Hello, sorry for the delay in answering. I'm including a summary of the paper, so you'll see what parameters were measured.

Regards,

World Veterinary Poultry Association Convention (WVPAC), September 16, 20, 2019, Bangkok, Thailand

Cellular immune response after vaccination with SG 9R in birds fed Aflatoxin and a mycotoxin absorbent in the diet.
1 A. Martins, 1 T. Furian, 1 V. do Nascimento, and 2 M. Contreras *
1 Federal University of Rio Grande do Sul, Brazil 2 Special Nutrients, Miami, Florida, USA
Keywords: SG9R, Aflatoxin, Salmonella, Adsorbent, Mycotoxins

Introduction
Most developed countries around the world have eradicated Salmonella Gallinarum and S. Pullorum from commercial flocks, and now concentrate mainly in the prevention of infections caused by the paratyphoid group. However, a high percentage of the commercial layer’s world population is still affected by S.G., therefore vaccination with a live attenuated vaccines of S. Gallinarum (SG 9 R) is a strategy frequently used in some areas. After vaccination, developing immunity is critical to get the right protection against field challenge. One serious factor affecting the development of immunity is the presence of mycotoxins, including Aflatoxin (AFL). The objectives of this experiment were threefold: measuring the immune response in birds vaccinated with SG9R, quantifying the impact of AFL on the immune response, and determining if the mycotoxin absorbent used had an effect on the immune response.
Materials and Methods
This experiment was conducted at Imunova Laboratory in the Federal University of Parana (UFPR) in Brazil. A total of 240-day-old commercial baby chicks (Hendrix®, ISA- Brown®) free of Salmonella, were raised in isolation units since day one, to avoid cross contamination among treatments. All birds had ad libitum access to feed and water. Pullets were divided randomly in 8 treatments of 30 birds each. T1 was the negative control (no vaccine, no AFL, no absorbent), T2 was vaccinated, intoxicated (2.5 ppm) and treated with the absorbent (2.5 kg.ton), T3 was only vaccinated, T 4 was vaccinated and intoxicated (2.5 ppm), T 5 was only intoxicated with AFL (2.5 ppm), T 6 intoxicated (2.5 ppm) and treated with the absorbent (2.5 kg/ton), T7 was vaccinated and intoxicated with AFL (1 ppm), and T8 was vaccinated, intoxicated (1 ppm) and treated with the absorbent (1 kg/ton).
The feed was analyzed to detect any possible contamination with Salmonella or Aflatoxin. SG 9 R, a Samonella Gallinarum live attenuated vaccine was bought from Merck Animal Heath, USA. AFL was obtained from LAMIC, Federal University of Santa Maria in Brazil and mixed with the feed using two inclusion rates. Mycoad®, the mycotoxin absorbent (clay) was added using two inclusion rates, and bought from Special Nutrients in Florida, USA. The immune response was measured with flow cytometry (FACSCalibur® /Becton Dickinson). The data obtained was analyzed with software from one the following companies: FlowJo® (TreeStar) or WinMDI 2.9 (Joseph, Trotter). Four types of T lymphocytes were measured with flow cytometry. Body weight and mortality were recorded during the duration of the trial and all birds were euthanized at the end of the trial. After post mortem sessions were performed, macroscopic changes were recorded and histopathological studies were performed at UFPR.
Immune response/Blood results. The statistical analyses were performed separating the results for each blood collection. All treatments were compared against each other. An analysis of variance (Anova) with the method of Fisher PLSD (P 0.05) comparing the media of all groups among themselves was done. StarView® (SAS Institute, Inc.) was the statitical program used for this evaluation.
Table 1. Summary of activities during the whole experiment.
Day Activity
1 Placement in isolation units
• Aflatoxin mixed with the ration (T2, T4, T5, T6, T7 and T8)
• Mycoad mixed with the ration (T2, T6, T8)
• Weigh birds
28 • Vaccination via subcutaneous injection with
SG 9 R (T2, T3, T4, T7, T8)
31 • Weigh birds
• Blood samples collection (26 birds/treatment)
41 • Blood samples collection (26 birds/treatment)
42 • Weigh birds
49 • Blood samples collection (26 birds/treatment)
• Birds euthanized (all)

• 0.2 ml of SG 9 R vaccine with a titer of 10 4 colony forming units (CFU) was injected subcutaneously

Results/Discussion
Performance. The control treatment (T1), vaccinated treatment (T3), and the vaccinated/intoxicated treatment treated with Mycoad-1 kg/mt (T8) showed the same statistically significant body weight during the whole experiment (table2). At 31 and 42 days, the treatment only intoxicated with AFL (T5), showed a significantly lower body weight than the control (T1), vaccinated treatment (T3), and the vaccinated/intoxicated treatment treated with Mycoad-1 kg/mt (T8). No mortality was reported in any of the groups. All intoxicated treatments not treated with the absorbent showed a significant reduction in body weight when compared to the non-intoxicated birds. This reduction in body weight was also reported in young commercial layers in a previous publication (4).
Table 2. Average body weight of commercial brown pullets at different ages during the trial.
Treatments
Age
Vaccine AFLA Absorbent 1 day 31 days 42 days
T1 - - - 42,2 a 415,7 a 505,1 a
T2 SG9R 2,5 PPM 2,5 Kg 42,84 a 316,6 b 444,7 a
T3 SG9R - - 41,3 a 371,7 a 483,2 a
T4 SG9R 2,5 PPM - 41,87 a 221,8 c 322,2 b
T5 - 2,5 PPM - 41,07 a 270,7 c 360,7 b
T6 - 2,5 PPM 2,5 Kg 43,39 a 338,3 b 422,5 b
T7 SG9R 1 PPM - 44,01 a 369,7 b 452,3 a
T8 SG9R 1 PPM 1 Kg 42,95 a 412,8 a 481,5 a
a ,b, c Values within one column with different letters are significantly different (P = 0.05).

Macroscopic observations. Vaccinated birds (T3) showed enlarged livers, slightly greenish with rounded lobules (picture 1). Birds intoxicated with AFL (T5) and birds intoxicated and vaccinated (T4, T 7) showed a fragile liver with a yellowish appearance and the presence of multiple red dots. Other groups didn’t show any lesions (picture 2).
Microscopic observations. Histopathological evaluation of the liver in T 5 showed the typical lesions produced by an intoxication with AFL, that is, lipidic vacuolization of the hepatocytes, areas of necrosis and hypertrophy of the biliary ducts.
Immune Response. This experiment shows a significant reduction in the concentration of helper T lymphocytes in the treatments intoxicated with AFLA. Peripheral helper T lymphocytes (table 3) and mucosal auxiliary T lymphocytes (table 4) showed a significant reduction when comparing the vaccinated treatment (T3) with the vaccinated and intoxicated treatment (T 4). A significant reduction in the concentration of cytotoxic T lymphocytes (tables 5 and 6) is observed at 42 days. This reduction is also observed when comparing the vaccinated treatment (T3) with the vaccinated and intoxicated treatment (T4). Other researchers (3) have reported that the inclusion of corn naturally contaminated with AFL and Fumonisin significantly decreased CD4 + and CD8 + T lymphocytes in the intestinal mucosa of broilers. Feeding naturally contaminated corn with Fusarium toxins, another report (2) found a reduction in white blood cells, B lymphocytes, CD4 + and CD8 + T lymphocytes.
Table 3. Percentage of peripheral auxiliary T lymphocytes (CD4+TCRVß1-) in blood.
Treatments
Blood collections
Vaccine AFLA Absorbent 31 days 42 days 49 days
T1 - - - 11,36 ± 8,99 a 7,38 ± 4,11 a 2,01 ± 1,17 a
T2 SG9R 2,5 PPM 2,5 Kg 1,45 ± 1,17 b 5,96 ± 1,72 a 1,64 ± 0,47 a
T3 SG9R - - 3,11 ± 0,98 a 4,99 ± 1,41 a 2,00 ± 0,83 a
T4 SG9R 2,5 PPM - 2,78 ± 2,23 b 3,11 ± 0,45 b 1,71 ± 0,50 a
T5 - 2,5 PPM - 11,91 ± 7,82 a 4,40 ± 1,34 a 1,37 ± 0,40 a
T6 - 2,5 PPM 2,5 Kg 5,44 ± 5,76 a 5,47 ± 2,07 a 1,22 ± 0,37 a
T7 SG9R 1 PPM - 3,10 ± 2,15 a 5,16 ± 2,84 a 1,26 ± 0,30 a
T8 SG9R 1 PPM 1 Kg 2,80 ± 1,89 a 4,33 ± 1,27 a 1,30 ± 0,51 a
Values within one column with different letters are significantly different (P = 0.05). Mean ± Standard Deviation.

Table 4. Percentage of mucosal auxiliary T lymphocytes (CD4+TCRVß1+) in blood.
Treatments
Collections
Vaccine AFLA Absorbent 31 days 42 days 49 days
T1 - - - 12,77 ± 5,86 a 0,92 ± 0,32 a 5,17 ± 3,20 a
T2 SG9R 2,5 PPM 2,5 Kg 7,69 ± 5,58 a 0,77 ± 0,32 a 3,46 ± 0,81 a
T3 SG9R - - 9,74 ± 6,34 a 0,66 ± 0,34 a 4,46 ± 2,22 a
T4 SG9R 2,5 PPM - 3,35 ± 0,91 b 0,46 ± 0,15 b 3,90 ± 1,21 a
T5 - 2,5 PPM - 4,22 ± 2,71 b 0,74 ± 0,28 a 4,85 ± 1,56 a
T6 - 2,5 PPM 2,5 Kg 3,87 ± 3,15 b 1,02 ± 0,42 a 3,81 ± 1,05 a
T7 SG9R 1 PPM - 4,59 ± 4,08 b 0,87 ± 0,55 a 3,86 ± 0,88 a
T8 SG9R 1 PPM 1 Kg 3,64 ± 3,26 b 0,75 ± 0,19 a 3,81 ± 1,46 a
Values within one column with different letters are significantly different (P = 0.05). Mean ± Standard Deviation.

Table 5. Percentage of mucosal cytotoxic T lymphocytes (CD4-TCRVß1+) in blood.
Treatments
Collections
Vaccine AFLA Absorbent 31 days 42 days 49 days
T1 - - - 2,61 ± 2,13 a 1,62 ± 0,58 a 3,11 ± 1,96 a
T2 SG9R 2,5 PPM 2,5 Kg 0,83 ± 0,61 a 1,23 ± 0,71 a 1,85 ± 0,56 a
T3 SG9R - - 2,15 ± 1,81 a 1,33 ± 0,62 a 2,33 ± 1,22 a
T4 SG9R 2,5 PPM - 2,38 ± 1,99 a 0,64 ± 0,17 b 1,71 ± 0,76 a
T5 - 2,5 PPM - 1,43 ± 0,99 a 1,25 ± 0,47 a 2,00 ± 0,69 a
T6 - 2,5 PPM 2,5 Kg 1,73 ± 1,03 a 1,15 ± 0,27 a 1,60 ± 0,76 a
T7 SG9R 1 PPM - 2,09 ± 1,52 a 1,28 ± 0,68 a 1,51 ± 0,26 b
T8 SG9R 1 PPM 1 Kg 1,71 ± 1,61 a 1,32 ± 0,42 a 2,48 ± 1,12 a
Values within one column with different letters are significantly different (P = 0.05). Mean ± Standard Deviation.

Table 6. Percentage of activated cytotoxic T lymphocytes (CD8+CD28-) in blood.
Treatments
Collections
Vaccine AFLA Absorbent 31 days 42 days 49 days
T1 - - - 9,53 ± 7,38 a 1,26 ± 0,97 a 2,53 ± 1,27 a
T2 SG9R 2,5 PPM 2,5 Kg 0,78 ± 0,68 b 1,11 ± 0,49 a 0,87 ± 0,27 b
T3 SG9R - - 2,24 ± 0,90 b 1,36 ± 0,97 a 1,00 ± 0,46 b
T4 SG9R 2,5 PPM - 1,51 ± 1,13 b 0,59 ± 0,15 b 1,38 ± 0,63 a
T5 - 2,5 PPM - 9,86 ± 5,80 a 2,00 ± 1,17 a 2,78 ± 1,21 a
T6 - 2,5 PPM 2,5 Kg 1,58 ± 1,72 b 1,14 ± 0,43 a 1,92 ± 0,62 a
T7 SG9R 1 PPM - 1,50 ± 1,52 b 1,31 ± 1,04 a 1,74 ± 0,56 a
T8 SG9R 1 PPM 1 Kg 1,08 ± 0,99 b 1,29 ± 0,49 a 1,86 ± 0,57 a
Values within one column with different letters are significantly different (P = 0.05). Mean ± Standard Deviation.

Conclusions
SG9R vaccination and the inclusion of a mycotoxin binder in birds vaccinated and intoxicated did not have a significant negative effect on body weight. SG9R vaccination promoted a statistically significant reduction in the concentration of activated cytotoxic Lymphocytes T (CD8-CD28-). This marker can be measured as a parameter before and after vaccination in commercial layers. AFL showed an important suppressive effect on mucosal auxiliary T lymphocytes (CD4+TCRVß1+), periferic auxiliary T lymphocytes (CD4+TCRVß1-), and mucosal cytotoxic Lymphocytes (CD4-TCRVß1+) in vaccinated birds. A significant difference (p?0,05) was detected in blood samples taken 3 and 14 days after vaccination. The inclusion of 2.5 kg/ton of the mycotoxin absorbent in feed neutralized the negative effect on the imune parameters already mentioned. A significant increase (p? 0,05) in the concentration of mucosal auxiliary T lymphocytes (CD4+TCRVß1+), auxiliary periferic T lymphocytes (CD4+TCRVß1-) and mucosal cytotoxic T lymphocytes (CD4-TCRVß1+) was detected at 42 days of age.

References:
1. Chang, C. F. and Hamilton P. B. Impaired phagocytosis by heterophils from chickens during aflatoxicosis. Toxicol Appl Pharmacol. 48, 459–66. 1978
2. Chowdhurry, S. R.; Smith, T. K.; Boermans, H. J.; and Woodward, B. Effects of feed-borne fusarium mycotoxins on hematology and immunology of laying hens. Poultry Science, v 84, p. 1841-1850, 2005.
3. Milglino, L.B et al. Efeito de Milho Naturalmente Contaminado com Micotoxinas sobre a Imunidade e Desempenho em Frangos de Corte. 2012. 63 f. Dissertação de Mestrado – Curso de Pós-graduação em Ciências Veterinárias, Universidade Federal do Paraná.2012.
4. Oliveira, C.A. et al. Produção e qualidade dos ovos de poedeiras submetidas à intoxicação prolongada com aflatoxina B1. Arquivos do Instituto Biológico, v.68, p.1-4, 2001.




Picture 1. Control (T1) vs. vaccinated birds (T3) at 49 days of age.

Picture 2. Treatments intoxicated with AFL (T4, T5, T7) , without the inclusion of Mycoad vs. treatments exposed to AFL and fed the absorbent (49 days of age).


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Manuel Contreras Manuel Contreras
Doctor of Veterinary Medicine
Special Nutrients Special Nutrients
Florida, United States
May 22, 2020

By comparing the group where Afla and the mycotoxin binder was used with the treatments where only Afla was added and the negative control (no Afla, no mycotoxin binder), you can see the effect of the binder tested on the immune response.

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