Total plasma proteins and hemoglobin levels as affected by worm burden in freshwaterfish in Morogoro, Tanzania

P S Mlay, M Seth, S T Balthazary, R T Chibunda, E C J H Phiri and O B Balemba

Department of Physiology, Biochemistry, Pharmacology and Toxicology,
Sokoine University of Agriculture, Morogoro, Tanzania
panejo@yahoo.com

Abstract

The present study was carried out to establish normal blood levels of hemoglobin and total plasma proteins and the impact of worm infestation on these parameters in freshwater fish under natural and artificial rearing systems in Morogoro, Tanzania. The standard cyanmethemoglobin and Biuret methods were used to determine hemoglobin concentration (Hb), and plasma total protein (TP) respectively; physical counts of grossly visible worms and larvae in the gastrointestinal tract and body cavity in 122 fish: Clarias gariepinus (catfish) (23), Oreochromis karomo (26), Oreochromis urolepis (30) and Oreochromis niloticus (tilapia) (43). Mean Hb concentrations were 9.7 g/dl for Clarias gariepinus, 7.5 g/dl for Oreochromis karomo, 7.6 g/dl for Oreochromis urolepis and 9.2 g/dl for Oreochromis niloticus.

Mean plasma total protein concentrations were 4.3, 3.6, 3.7 and 3.8 g/dl respectively for the above fish species. Catfish had higher (P<0.05) mean blood Hb and plasma total protein compared to tilapia fish. Whereas, tilapia fish were free of grossly visible worms, catfish were infested with Anisakis spp (mean 12.8; range 2-36 larvae/fish). The worm burden was negatively correlated with blood Hb (R= -0.33) and total plasma proteins (TP) (R= -0.37).

With tilapia and catfish in the same macro-environment, Anisakis spp, which is zoonitic selectively infest catfish. Our results show that fish species, sex, the habitat and worm infestation influence hematological parameters. In addition, we have uncovered the potential for zoonosis by Anisakis spp in freshwater catfish in Morogoro, Tanzania suggests for a need to investigate freshwater fish parasites in the region.

Keywords: Anisakis spp., catfish, freshwater fish, hemoglobin, plasma proteins, Tilapia

Introduction

Fish is an important source of protein to humans and other animals. Fish industry also offers employment opportunities to many people as well as income at household and national levels (FAO 1996; Srivastava 1988). Due to the rapid rise in human population, there is tremendous pressure on natural fish resources, which are on the decline (FAO 1996). Thus, small to large-scale fish farming is on the increase as an attempt to increase fish availability to meet the ever-increasing protein demand for rising human populations (Satchell 1991; Mwangulumba 1997 unpublished report). In order to maximize fish productivity, farmers need to be aware of the factors that influence fish performance such as nutrition, diseases, environmental stresses and pollutants (Lebelo et al 2001).

The health of fish can be affected by environmental factors (stress), nutrition as well as by pathogens. Stress in fish may be induced by various abiotic environmental factors such as changes in water temperature, pH, oxygen concentration and water pollutants including pesticides, insecticides (Meier et al 1983; Lebelo et al 2001), petroleum products and heavy metals (Witeska 2005). Biotic interactions such as predator pressure, parasitic invasions or strong competition with other organisms or among the fish in overcrowded areas and by human activities related to fish rearing and harvesting (manipulation, transport) can also be a source of stress to fish (Witeska 2005). Stress reaction involves various physiological changes including alteration in blood composition and immune mechanisms. Stress induces also changes in blood cell numbers and activities. An increase in hematocrit, red blood cell count and volume, and hemoglobin level usually has been reported to fish subjected to stress (Wendelaar Bonga 1997). The increase in the number of circulating RBC is thought to be associated with the release from reservoirs (spleen contraction) and even division of circulating cells in fish subjected to low oxygen tension (Murad et al 1993). A decrease in white blood cell count, especially of lymphocytes usually occurs in fish subjected to stress (Elsaesser and Clem 1986). However, the level of phagocytes sometimes increases with decreased activity of lymphocytes and phagocytes (Elsaesser and Clem 1986). Heavy metal toxicity invariably reduces white blood cell count particularly lymphocytes (Witeska 2005) leading to compromised immune responses in the affected fish.

Fish parasites are of economic importance in that they affect the productivity of fish through mortalities, by decreasing growth rate, efficiency in feed conversion ratio and levels of the total plasma proteins due to a fall in absorbed amino acids that are essential for protein synthesis as well as lowering the quality of the meat (Fraser and Mays 1986). Nematodes, cestodes and trematodes helminthes are common in both wild and cultured fish. Fish frequently serve as intermediate or transport hosts for larval parasites of many animals, including humans (Fraser and Mays 1986). Larval migrations of fish helminthes e.g., Proteocephalus ambloplites, have been associated with reproductive failure in fish. Occasionally heavy infestations of intestinal worms have been associated with mechanical obstruction of the lumen of the gut extensive damage to intestinal mucosa leading to enteritis and anemia (Fraser and Mays 1986). The aim of this study was to investigate the blood levels of hemoglobin and total plasma proteins and the impact of worm infestation on these hematological parameters in freshwater fish under natural and artificial rearing systems.

Materials and methods

Study area

The study was carried out in the Morogoro urban and peri-urban areas. Morogoro town is located along Latitude 6⁰ 51^' South and Longitude 31⁰ 41^' East at an altitude of 530 m above the sea level. Samples of fish used in this study were taken from Mindu dam that is located about 8 kilometers west of Morogoro Municipality along the highway to Iringa and Mnembuka's Levee pond, in Kihonda ward situated about 5 km from town center along Dodoma highway.

Mindu dam covers an area of about 10 km² and is surrounded by Uluguru Mountains from which a good number of rivers feed the dam. The dam was constructed primarily as a source of water for Morogoro urban population and currently it supplies water to about 70% of the people in the Morogoro Municipality and is also a source of fish to people in Morogoro. Mnembuka's Levee pond was constructed using concrete walls and floor with dimensions of about 6 m x 3 m x 2 m (length x width x height). It is an aquaculture pond hence fish live in an artificial condition unlike those in Mindu dam. The fish depend on aquatic organisms as well as supplements like maize bran.

Fish species

Fish of genus Oreochromis (Tilapia); mainly Oreochromis karomo and Oreochromis urolepis, Oreochromis niloticus and Clarias gariepinus (Catfish) were used in this study. Samples from Mindu dam comprised of both tilapia (56) and catfish (23) while Kihonda samples were tilapia only (43). The type, sex and number of fish sampled from the two ecosystems are indicated in Table 1. Fish from either source were caught early in the morning, transferred into plastic buckets (5-6 fish/bucket) containing 20 litres water and quickly transported to the laboratory.

Blood sample collection

In the laboratory, anaesthetize using a solution of tricane methanesulphate (MS 222) at 62.5mg/litre (Lebelo et al 2001) after which the fish were carefully wiped dry and put on lateral recumbence. A 23 gauge needle was inserted into the caudal vein using the lateral line as a landmark. Approximately 2-5 ml of blood was collected from each fish. Each blood sample was divided into two portions int-o tubes containing heparin as an anticoagulant. One portion was used for determination of hemoglobin concentration while the second portion was centrifuged at 12,000xg for 10 minutes to obtain the plasma, which was then extracted into sterile 2 ml vials then stored at -20 ^°C up to the day of analysis for total plasma proteins.

Hemoglobin concentration (Hb)

Hemoglobin was measured using the standard cyanmethemoglobin method described by Baker and Silverton (1976). Blood sample (20 ml) was diluted in 5 ml of the diluent in a test tube and thoroughly mixed using a vortex mixer. The absorbance of the solution was measured by spectrophotometer (Cecil 2000 spectrophotometer, UK) after 10 minutes of standing time at a wavelength of 540 nm. The absorbance of cyanmethemoglobin was then used to obtain hemoglobin concentration (in g/dl), using a standard Hb estimation chart.

Total plasma proteins (TP)

The total plasma proteins were measured by using the standard Biuret method as described by Lawrence (1986), which is based on the reaction between the peptide bonds of protein and Cu²⁺ (from copper sulfate solution) that produces a blue-violet colored complex in alkaline solution. The measurements were done using the Biuret method (CHRONOLAB) where 100 ml of blood plasma and standard protein solution were diluted into 500 ml of the Biuret reagent in a test tube. The Biuret reagent without a sample being added was used as a blank. After mixing the test tubes were left to stand for 30 minutes and thereafter the absorbance was read using spectrophotometer (Cecil 2000, UK) at a wavelength of 540 nm.

The calculation of the total proteins was done using the following formula.

From the ratio:

The values of total plasma proteins obtained were expressed in g/dl

Gastrointestinal worms

Determination of gastrointestinal worms was based on establishment of the number and types of grossly visible worms. Briefly the anesthetized fish had their abdominal cavity opened by cutting along the sides of the abdomen to expose the viscera. The macroscopically visible worms found within the abdominal cavity as well as those found inside the stomach and intestinal lumen were collected, counted and preserved in 20 ml universal bottles containing 15 ml of 70% alcohol. The identification of worms was done in the Department Veterinary Microbiology and Parasitology, Sokoine University of Agriculture.

Statistical analysis

Statistical analysis was done using SAS (1988) method and model:

Y= I + T + C + S+ ε

Where

Y= dependable variable

I= Intercept

T = Fish type (Catfish or Tilapia)

C = Fish source (Mindu or Kihonda)

S = Sex (Male or Female)

ε = Random error

Results

Body parameters

The weight, length and girth of the sampled fish are shown in Table 2. Mindu fish were generally heavier and larger compared to Kihonda Levee fish.

Hemoglobin concentration

The hemoglobin concentration of Mindu catfish ranged from 6.0-16.7 g/dl with raw mean value of 9.7±2.6 g/dl (Table 3).

Male catfish had higher Hb mean values compared to females 10.7 g/dl Vs 8.0 g/dl which was significant (P<0.01) (Table 4).

Kihonda tilapia had higher Hb values compared to Mindu Tilapia while TP levels similar. The Hb concentration of Kihonda tilapia and Mindu catfish did not differ (Table 3). Mindu Catfish had higher values for both Hb and TP compared to Mindu tilapia as well as higher TP compared with Kihonda tilapia and the difference was significant (P<0.05) (Table 5).

The effects of sex and species on the Hb and TP are shown in Table 6. Male fish had higher values compared to females particularly with respect to the Hb values of Clarias gariepinus and Oreochromis karomo. However, female Oreochromis urolepis and Oreochromis karomo had higher TP compared to the males of the same species.

Total plasma proteins (TP)

Row and least square mean values of Hb and TP of Mindu catfish and Tilapia fish of both sexes from the two sources are shown in Tables 4 and 6, respectively. The TP concentration did not differ between male and female (4.6 Vs 4.0 g/dl, respectively)(Table 4).

Fish helminthes

Almost all catfish were infested with helminthes belonging to the Anisakis spp with the numbers of macroscopically observable larvae ranging from 3 to 36 per fish (Table 3). The number of worms was poorly and negatively correlated to both the total plasma proteins (R= -0.37) and blood hemoglobin level (R= -0.33). Interestingly, tilapia fish from both sources (Mindu dam and Kihonda levee pond) were free of grossly visible gastrointestinal worms
 

Discussion

In the present study, we found that factors influencing hematological parameters thus, fish productivity in freshwater rearing in Morogoro, Tanzania included: fish species, sex, worm infestation rate and the habitat. Anisakis spp was the only type of helminthes that was found in fish in Mindu dam. Although catfish and tilapia were reared in Mindu dam, Anisakis spp selectively infested catfish alone suggesting that tilapia fish are resistant to these helminthes. This supports the findings by Balfry et al (1997) that there are strains and species differences in diseases resistance in fish.

The mean hemoglobin concentration for the catfish was within the range reported in other studies (Larsson et al 1976; Satchell 1991; Lebelo et al 2001) though it was higher than that reported by Hattingh (1972) in the South African Clarias gariepinus (5.8 g/dl). This deviation suggests that geographical location (temperature, type of feed/nutrients) and gradual changes in the environment, fish physiology and genetics with time may have influence on the Hb of catfish supporting the concept that Hb is greatly influenced by environmental factors such as feed/nutrient and oxygen availability, presence of toxic ingredients and other stress factors (Srivastava 1988; Luskova 1998). Higher Hb content and worm infestation in catfish may be explained by behavioral differences and habitat preferences since catfish prefer muddy waters while tilapia spend most of their time in clear surface waters (Lowe-McConnell 1975). Muddy waters have low dissolved oxygen and worm larvae and eggs compared to surface clear water. Low oxygen tension necessitates elevation of hemoglobin concentration per volume of blood in order to keep gaseous exchange going on optimally (Murad et al 1993; Lebelo et al 2001). Presence of worm eggs and larvae and crustaceans that act as intermediate host for many helminthes in muddy waters is the most likely factor leading to greater chances of worm infestation among catfish and not tilapia fish (Lowe-McConnell 1975). However, whether tilapia fish are resistant to Anisakis spp or not cannot be ruled out.

The significantly higher Hb values in male compared to female catfish are consistent with the literature (Larsson et al 1976). However, during spawning periods females tend to have higher blood cell count especially red blood cells, causing increased hemoglobin concentration and the increase has been proposed to be caused by elevation of estrogen hormone, which induces increase of erythropoietin hormone activity (Eisler 1965; Luskova 1998). The observation that Kihonda tilapia had higher Hb values compared to Mindu Tilapia and that and TP values of tilapia from either source did not differ suggest that Hb values are more affected by the microenvironment compared to TP. Alternatively, this difference could be attributed to differences in the nutritional status as Kihonda fish were supplemented with maize bran. Data of the total plasma proteins for catfish and tilapia obtained in this study with catfish having non-significantly higher values compared to tilapia are within the reported range (Sulya et al 1961; Larson et al 1976; Schaperclaus 1991; Lebelo et al 2001). Although the overall impact of worm burden to plasma proteins and blood hemoglobin was not evident, a low worm burden was associated with high levels plasma proteins and blood hemoglobin (Lawrence 1986). When the worm number is high there is increased blood loss caused by hemorrhage and consumption by worms; leading to an overall low Hb. This can affect the productivity of the fish through mortalities, by decreasing growth rate, reducing the quality of the meat and making the hosts more susceptible to pathogenic parasites and bacteria.

The presence of Anisakis spp in freshwater fish in Morogoro is of epidemiological significance and urge for the need to control this parasite in the dam. Not only that heavy worm infestation leads to poor performance in fish (Hiscox and Broksen 1973) but Anisakis spp are zoonostic and cause severe intestinal damage in humans infested by eating raw fish (Roberts and Shepherd 1974).

Fish from Mindu dam were generally bigger compared to those from Kihonda Levee pond. The difference is likely due to the fact that the fish from Kihonda Levee pond were frequently harvested from the pond, which is smaller in size based on family needs compared with Mindu dam, which is larger.

In conclusion species, ecological habitat, and sex and health status influence hematological parameters in fish. The Hemoglobin and total plasma proteins levels obtained in this study were within the range reported elsewhere. In most cases, male fish had relatively higher values compared to female fish. Also, the extent of worm infestation observed in this study did not have any significant impact on the levels of total plasma proteins and hemoglobin. This is the first report of Anisakis spp zoonosis in freshwater fish in Tanzania.

Acknowledgement

The authors would like to express sincere thanks to Dr. Mnembuka, B. V. for allowing free access to fish reared in his pond and Mr. Kibirige, W. K. B. and Jingu, P. K. for technical support during this study. We acknowledge fishermen at Mindu for co-operation. We also acknowledge the Government of Tanzania for financial support through Sokoine University of Agriculture.

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Received 22 August 2006; Accepted 25 September 2006; Published 8 February 2007

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