**3. Results**

The effects of *Lactobacillus acidophilus* as a supplement on gut microbiota of *Macrobranchium vollenhovenii* are presented in **Table 2**. The total viable bacteria count (TVC) was significantly decreased in the gut samples of prawn fed with feed supplemented diets (p < 0.05). Similarly, total enterobactereceae (TEB) was reduced significantly in fish fed fortified diets. The highest TVC and TEB were recorded in fish fed control diet.

**Table 3** depicts microbiota composition of *Macrobranchium vollenhevenii* fed diets fortified with *Lactobacillus acidophilus*. The results indicated the species composition of the prawn varied significantly with respect to levels of inclusion of *Lactobacillus acidophilus.* The dominated species were *Pseudomonas aeruginosa, Aeromonas hydrophila, Bacillus and Staphylococcus* species.


Means (Log10 cgu/mL) with different superscripts are significantly different (p < 0.05), while, absence of letters means no significantly different (P > 0.05). TVC = Total viable bacteria count; TEB = Total enterobacterceae.

**Table 2.** Gut microbiota of *Macrobranchium vollenhovenii* fed diets fortified with *Lactobacillus acidophilus*.

room temperature. After incubation, the supernatant removed from the plate and NBT reduction fixed with 100% methanol for 10 min. The plate was then washed with 70% methanol, and left to air dry. A mixture of 120 mL of 2 M potassium hydroxide and 140 mL DMSO was added to dissolve the resulting formazan blue crystals. The NBT reduction was measured using the microplate reader (Optica, Mikura Ltd., UK) at 630 nm, and respiratory burst activ-

Total haemocyte count (THC) was performed in a haemocytomter using microscope. The phenolosidase activity (PO) was evaluated by measuring the formation of dopachrome L-dihydrophenylclamine (L-DOPA) at 490 nm with the aid of spectrophotometer. While

(dependent oxidation of phenol red) while chemiluminescence was used to measure the light emission from reactive oxygen intermediates [18]. Lysozyme activity of fish sera was determined by using lysoplate technique [19]. In brief, 0.60 mg/mL *Micrococcus luteus* was cast in 1% agarose gel (Difco, USA) with 50 mM phosphate buffer (pH 6.2). Wells (6 mm) were created nutrient agar plates and were filled 25 μL of serum samples and incubated for 20 h at 25°C. Lysozyme activity was calculated from a standard curve prepared with lysozyme from chicken egg white. The respiratory burst activity was measured using diagnostic reagent kits (Randox, London, UK) as described by Chiu et al. (2007). Relative protection level (RPL) was estimated as RPL = [(1−%mortality in treatment)/% mortality in control) ×

The results were presented as mean ± SE of three replicates. Prior to statistical analysis, all data were tested for normality of distribution using the Kolmogorov–Smirnov test. The homogeneity of variances among different treatments was tested using Bartlett's test. Then, data were subjected to one-way ANOVA to evaluate effects of *Lactobacillus acidophilus* supplementation. Differences between means were tested at the 5% probability level using Duncan test. The optimum *Lactobacillus acidophilus* level was determined using polynomial regression analysis. All the statistical analyses were done using SPSS program version 20 (SPSS, Richmond, VA, USA).

The effects of *Lactobacillus acidophilus* as a supplement on gut microbiota of *Macrobranchium vollenhovenii* are presented in **Table 2**. The total viable bacteria count (TVC) was significantly decreased in the gut samples of prawn fed with feed supplemented diets (p < 0.05). Similarly, total enterobactereceae (TEB) was reduced significantly in fish fed fortified diets. The highest

**Table 3** depicts microbiota composition of *Macrobranchium vollenhevenii* fed diets fortified with *Lactobacillus acidophilus*. The results indicated the species composition of the prawn varied significantly with respect to levels of inclusion of *Lactobacillus acidophilus.* The dominated species were *Pseudomonas aeruginosa, Aeromonas hydrophila, Bacillus and Staphylococcus* species.

TVC and TEB were recorded in fish fed control diet.

O2

by horseadish peroxidase

ity was expressed as NBT reduction.

98 Aquaculture - Plants and Invertebrates

100] [20].

**3. Results**

**2.6. Statistical analysis**

reactive oxygen intermediates (ROI) were used to measure H2


Means (Log10 cgu/mL) with different superscripts are significantly different (p < 0.05), while, absence of letters means no significantly different (P > 0.05).

**Table 3.** *Gut microbiota composition of Macrobranchium vollenhovenii fed diets fortified with lactobacillus acidophilus*.

*Pseudomonasa aeruginosa and Aeromonas hydrophila* were reduced significantly (p < 0.05) while *Bacillus* species was greatly increased. However, there was no significantly difference in the load of *Staphylococcus* species. The highest *Pseudomonas aeruginosa, Aeromonas hydrophila* and *Staphylococcus* species were recorded in prawn fed control diet while the least was observed in prawn fed diet fortified with 10<sup>5</sup> cfu/mL *Lactobacilus acidophilus*. However, the highest *Bacillus* species was obtained in prawn fed diet supplemented with 105 cfu/mL *Lactobacilus acidophilus.*

Effects of *Lactobacillus acidophilus* based diets on innate immune responses of *Macrobranchium vollenhovenii* was presented in **Table 4**. The immune response of the prawn was stimulated by *Lactobacillus acidophilus* supplementation. The SOD, CAT, RBA, THC, PO, ROI and lysozyme activity were significantly different (p < 0.05) among the treatments. Highest SOD, CAT, RBA, THC, PO, ROI and lysozyme activity were recorded in prawn fed 10<sup>5</sup> cfu/mL diet; meanwhile, the lowest values were obtained in the control group.


Means with different superscripts are significantly different (p < 0.05), while, absence of letters means no significantly different (P > 0.05).

**Table 4.** Innate immune response parameters of *Macrobranchium vollenhovenii* fed diets fortified with *Lactobacillus acidophilus*.

**4. Discussion**

different (P > 0.05).

infection.

pressed the growth of pathogenic bacteria.

The result of the gut flora revealed that there were reductions in the bacteria load of the prawn fed fortified diets. The decrease in the in observed could be attributed to the activities of *Lactobacillus acidophilus* as probiotics which modulate the gut pH to its favour and outcompete the pathogenic organisms. According to International Commission on the Microbiological Specification of Foods [21] the acceptable level of bacterial load in fish tissue should be lower than 5.70 Log10cfu/g. In this study, the TVC and TEB of prawn fed fortified diets were within the recommended values except the control and fish fed 10<sup>1</sup> cfu/ mL of *Lactobacillus acidophilus*. The reduction in the load of bacteria especially TEB signifies that the probiotics improve the gut of the prawn by outcompete the pathogenic bacteria. The gut flora comprises *Pseudomonas aeruginosa, Aeromonas hydrophila,* Bacillus species and *Staphylococcus* species. Fish fed *Lactobacillus acidophilus* based diets had higher gut flora dominated by *Bacillus* species that the fish fed control diet and reduction of pathogenic organisms were observed. The findings of this study are in agreement with the work of [22] who reported that probiotics enhanced the population of beneficial bacteria and sup-

Means with different superscripts are significantly different (p < 0.05), while, absence of letters means no significantly

**Table 6.** Resistant of *Macrobranchium vollenhovenii* fed diets fortified with *Lactobacillus acidophilus* to *Aeromonas hydrophila*

**Initial stock Survival (%) Relative protection**

http://dx.doi.org/10.5772/intechopen.78010

101

Gut Microbiota and Innate Immune Response of *Macrobrachium vollenhovenii* Infected…

*Lactobacillus acidophilus* **inclusion levels (cfu/mL) Parameters (log10cfu/mL)**

Control 30 10.0 ± 0.01a 0.00 ± 0.00a 30 30.0 ± 0.01b 22.22 ± 0.03b 30 36.7 ± 0.15b 29.63 ± 0.11b 30 83.3 ± 0.21 81.48 ± 0.16 30 90.0 ± 0.13 88.89 ± 0.80 30 93.3 ± 0.04 92.59 ± 0.51

Gut flora have continuous and dynamic effect on the host's gut and systemic immune systems. The bacteria are key in promoting the early development of the gut's mucosal immune system both in terms of its physical components and function. The bacteria stimulate the lymphoid tissue associated with the gut mucosa to produce antibodies to pathogens [23]. The immune system recognises and fights harmful bacteria, but leaves the helpful species alone, tolerance developed in juveniles [24]. Recent findings have shown that gut bacteria play a role in the expression of toll-like receptors (TLRs) in the intestines, molecules that help the host repair damage due to injury. The TLRs are one of the two classes of pattern-recognition receptors (PRR) that provide the intestine the ability to discriminate between the pathogenic and commensal bacteria [25]. These PRRs identify

In addition, the exposure of prawn fed fortified diets to *Pseudomonas aeruginosa* infection was presented in **Table 5**. The result indicated that there was significant difference (p < 0.05) in survival rate and relative protection of prawn fed fortified diets. Highest survival rate and relative protection were recorded in group fed 105 cfu/mL *Lactobacilus acidophilus* inclusion level and least were recorded in control group.

**Table 6** reveals the resistant of *Macrobranchium vollenhovenii* to *Aeromonas hydrophila* infection. Survival and relative protection of prawn to *Aeromonas hydrophila* were significantly different (p < 0.05). Prawn fed 105 cfu/mL *Lactobacilus acidophilus* inclusion level had highest survival and relative protection rates while the prawn treated control diet had the lowest survival and relative protection rates.


Means with different superscripts are significantly different (p < 0.05), while, absence of letters means no significantly different (P > 0.05).

**Table 5.** Resistant of *Macrobranchium vollenhovenii* fed diets fortified with *Lactobacillus acidophilus* to *Pseudomonas aeruginosa* infection.


Means with different superscripts are significantly different (p < 0.05), while, absence of letters means no significantly different (P > 0.05).

**Table 6.** Resistant of *Macrobranchium vollenhovenii* fed diets fortified with *Lactobacillus acidophilus* to *Aeromonas hydrophila* infection.
