PRELIMINARY STUDIES ON THE EFFECTS OF ANIMAL
MANURE ON BACTERIAL DISEASE OF FISH

by

Ding Jinyi
Guo Xianzhen
Fang Xiuzhen
and Liu Meizhen

Asian-Pacific Regional Research and Training
Centre for Integrated Fish Farming
Wuxi, China

NACA Head Office
National Inland Fisheries Institute
Kasetsart University Campus
Fangkhen, Bangkok
Thailand
Cable: NACABK BANGKOK 10900

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INTRODUCTION

Although China has a long history of utilizing animal manure for fish farming, little is known about its effect on environmental sanitation. It is well known that animal manure fertilizes pond water and helps increase fish production. However, it may give rise to pathogenic microorganisms and parasites and cause water pollution. It has been observed that the use of animal manure increases the possibility of transfer of the parasites of pathegenes to humans. Studies have been undertaken to observe the relationship between fish-livestock farming system and the health of human beings in the Philippines and the United States, but little is known about the effects of animal manure on bacterial diseases in fish. Freshwater fish in China suffer most commonly from bacterial diseases such as Albinoderma, Erythroderma, Furunculosis, Stigtomatosis and verticle-scale disease, caused by Aeromonas and Psecudomonas. This study deals deals mainly with the possible effect of manure application : on bacterial diseases of fish particularly caused by Aeromonas sp. and Pseudomonas sp. Also observations were made on the occurrence of these bacteria in the manure, in pond water and in fish skin mucus.

MATERIALS AND METHODS

This experiment was mainly conducted at Helei Fish Farm, Helei Fishery Team No. 1 of Wuxi and Experimental Fish Farm of the Centre in five earthen ponds. Pond 1 was applied with chicken manure, Pond 2 with duck manure, Pond 3 with cow manure, Pond 4 with pig manure and pond 5 served as control without manure application.

Pond area. Pond 1, 2, 3, 4, 5 measured 0.34, 2.15, 2.15, 0.52 and 0.14 ha respectively. The water depth of all ponds ranged from 2–2.5 m. Table 1 illustrates fish species, stocking density, manuring rate and feeds used in the experiment.

Manure source. Chicken manure was obtained from our Centre's farm and pig manure come from the pigstyes near Pond 4 in Helei Fishery Team No. 1. Cow manure and duck manure were from Helei Fish Farm.

Sampling. From April 1985 to September 1986, chicken, duck, cow and pig manure and pond water were sampled seven times. On the other hand, B.C., G.C., S.C., B.H. were randomly sampled three times. All the samples were collected with standard sterilization techniques.

Manure samples. About 15–20 mg manure was collected and put into a sterilized dark bottle. About 0.5 mg manure sample was diluted with 49.5 ml normal saline water; this sample was cultured in flasks.

Water samples. About 125 ml water was collected randomly from fish ponds and 5 ml water from the sample was diluted with 45 ml normal saline water and were cultured in flasks.

Fish samples. Three individuals of B.C, G.C, S.C, B.H. were randomly sampled each time; 0.5 mg of mucus scraped from fish skin of each species was mixed and diluted with 49.5 ml normal saline water and cultured in flasks. Mucus was collected by scraping with sterilized cotton from an area of 30 cm² of each fish skin. Then, the average weight of mucus from those fishes was converted into the number of aerobic heterotrophic bacteria from 1 cm² fish skin according to the following formula:

Manure, water, fish mucus samples were diluted and cultured with nutrient medium. Aeromonas and Pseudomonas strains were identified through Gram staining, flagellum staining, observation of bacteria body, test of gelatic, alcohol oxidization and other bio-chemical reactions.

Calculation. Samples were cultured with nutrient medium for 43 hours at the temperature of 27°C. Then, the total number of aerobic heterotrophic bacteria was calculated in each sample.

RESULTS

The total number of aerobic heterotrophic bacteria in animal manure, pond water, fish samples are shown in Table 2.

Table 2 shows that the total number of aerobic heterotrophic bacteria in chicken, duck, cow and pig manure ranged from 10-10⁹ /g. Among these animal manure, chicken manure had the highest amount of aerobic heterotrophic bacteria followed by duck manure, pig manure and cow manure in subsequent order. In general, aerobic heterotrophic bacteria increased after manure application. In the experimental ponds, the number of aerobic heterotrophic bacteria in pond water was around 10³ - 10⁴ /ml. and about 10² - 10³ /ml in the control pond.

Table 1. Fish species, stocking density and manuring rate*

B.C. = black carp
G.C. = grass carp
S.C. = silver carp
B.H. = bighead carp
W.F. = wauchang fish
C.C. = common carp
CR.C. = crucian carp

Table 2. Aerobic heterotrophic bacteria in animal manure and pond water

Unit: cell/ml, cell/g

The weight flash skin mucus from B.C. G.C. S.C. and B.H was 0.17, 0.14, 0.1 and 0.18 g/30 cm² respectively. Table 3 illustrates the total number of aerobic heterotrophic bacteria in fish mucus per cm² which has been calculated from the number of aerobic heterotrophic bacteria in fish skin per gram. The aerobic heterotrophic bacteria from fish mucus in Pond 3,4 was (10³ -10⁵ /cm²), Pond 1, 2, (10²–10⁴). The difference of the aerobic heterotrophic bacteria among the manure-leveled ponds appears to be related to the amount of application of animal manure. During the experiment, manuring rate was higher in Pond 3 and 4, and the number of aerobic heterotrophic bacteria in fish mucus from the two ponds was found to be comparatively higher. (Table 3)

As B.C. G.C. S.C. and B.H. had different habitats, the number of aerobic heterotrophic bacteria in their mucus per cm² skin was also different (B.C. and G.C., (10³ - 10⁵ and S.C. B.H., 10²–10⁴).

Aeromonas sp. and Pseudemonas sp. were found to be present in all animal manure samples, pond water and fish skin mucus (Table 4).

Of 283 bacterial strains found in animal manure, pond water and fish mucus, 50 Aeromonas sp. and 28 Pseudemonassp. were identified. These two genera occurred nearly in very sample of animal manure and pond water. Aeromonas sp. were less in chicken, duck, cow and pig manure, but more in water of manured ponds. Among the manured ponds, Aeromonas sp. in Pond 2 had the highest incidence, followed by pond 1, 3 and 4 in subsequent order. Pseudomonas sp. occurred in animal manure and in pond water more or less in the same degree. However, both Aeromonas and Pseudomonas occurred more in water of manured ponds than in control pond.

Table 3. Distribution of aerobic heterotrophic bacteria in fish skin mucus

Pseudemonas sp. were present in the skin of fish from all the ponds. However, higher number of Pseudemonas sp. on B.C. AND G.C. (which were mid-water layer and bottom feeders) may be related to the higher incidence of aerobic heterotrophic bacteria on their skin.

Table 6 shows the fish production rates in different ponds.

Table 4. Occurrence of Aeromonas spp. and Pseudemonas spp in animal manure and pond water

DISCUSSION

Experimental results indicated that aerobic heterotrophic bacteria in chicken, duck, cow and pig manure ranged from 10 ⁷ -10⁰ /g (Table 2). However, Aeromones sp. and Pseudomonas sp. were less in animal manure used. Particularly, Aeromonas sp. was less in chicken, duck, cow and pig manure. Therefore, it appeared that relatively less amount of Aeromonas sp. and Pseulomonas sp. were brought into fish ponds with these animal manure. However, Aeromonas heterotrophic bacteria in the manured ponds were relatively more than those in control ponds. Following manure application a large number of bacteria grow and at the same time, Aeromonas sp. and Pseudemonas sp. increased in number, Aeromonas sp. in manured ponds in particular was higher than that in control pond (Table 4). Thus, it appeared that manure application had same effects on the growth of pathogenic bacteria which could cause fish diseases.

The amount of bacteria on fish skin is closely related to the bacterial biomass in the water column. With the input of manure and decomposition of organic matter at pond bottom, bacterial biomass increases from upper to bottom water layers. In the experiment, the aerobic heterotrophic bacteria in the mucus from 1 cm² fish skin differed from that of various fish species which had different habitats. Results indicated that the rate of aerobic heterotrophic bacteria in the skin mucus of mid and bottom water-layer fishes was higher than that of upper water-layer fish (B.C, 10³, S.C. B.H. 10²–10⁴). This evidence might indicate why B.C. and G.C. were acceptable to Erythoderma.

In summary the results show that while animal manure does not directly cause bacterial diseases in fish, the rate of pathogenic bacteria on fish skin has a close relationship with that in the pond water. Should the fish resistance to disease be low, there is the possibility of the occurrence of bacterial disease. Therefore, proper pond management should be observed to prevent bacterial disease, such management procedures to include fermentation of manure before its use, as well as ensuring sufficient food supply, good water quality, and maintaining the proper population and just the right the amount of manure input.

ACKNOWLEDGEMENT

The authors wish to express thanks to the International Development Research Centre (IDRC) for the great support of finance and research facilities. We are also greatly indebted to Dr. F. Brian Davy and Mr. Chen Foo Yan, NACA Coordinator for their keen interest in this work.

REFERENCES

1. Handbook of Fish Diseases, 1983 (In Chinese)

2. Wang Damin, 1977. Classification of Bacteria (in Chinese)

3. Sanitary Science, 1981 (in Chinese)

4. Xu Bohai et. al. 1980. Studies on the Stigmatosis of silver carp and bighead carp, Oceanologla Et Limnologia Sinica, Vol. 11. No. 1.

5. Wang Damin, 1956. Preliminary Studies on the Pathogenic Bacteria of Albinoderma on Black carp (in Chinese)

6. T. Rice et. al. 1984. Microbial Pathogens and Human Parasites In An Animal Waste Polyculture System. Progressive Fish Culturist 46: 230–238.

Table 5. Occurrence of Aeromonas sp. and P. Pseudmonas sp. in fish skin mucus.

Table 6. Fish production in manured and control ponds.