The present study aims to assess the epidemiology of fish parasites in cultured-fish populations. This study was carried out during a three year period (2005-2007) at a tench (Tinca tinca L.) fish farm located in the Northwestern region of Spain. 114 fish as well as 14 physicochemical and microbiological water parameters were analyzed in order to determine the main impact of both the host and the environmental parameters on the presence and the abundance of parasites in their host (host-parasite relation).Four different parasites, namely Ichthyophthirius multifiliis, Tripartiella sp., Myxobolus sp. and Gyrodactylus sp., were isolated, being the ciliates, with up to 100% of prevalence, the most abundant, with only 5.56% and 2.63% of the fish infected by the myxozoan and the monogenean, respectively. With the exception of the high alkalinity registered at the fish farm (pH > 9.0), the rest of the values fell within the recommended water quality parameters for cyprinids. The statistical and epidemiological analysis revealed that size, somatic condition and sex (intrinsic parameters) in addition to several environmental factors, linked to seasonality and poor water quality, have significant effect on the host-parasite relationship, increasing the likelihood of the parasite presence (p<0.050). We conclude that, in temperate climates, the development, reproduction and transmission of the parasites are triggered under the most stable conditions of the ecosystem (hydrological, physicochemical and biological) i.e. the warmer periods of the year, when the cyprinid populations display a more active and gregarious behavior. The results of the present study will also help understanding both the epidemiology and the parasitology of fresh water cultured-fish, and, what is more important, will help designing management strategies and procedures for preventing parasitic diseases.
Tench (Tinca tinca L.) is a fresh and brackish-water fish species of family Cyprinid of Eurasian distribution. In the Iberian Peninsula, it is widely distributed throughout all the river basins, mainly in shallow and lentic aquatic environments, though its populations are rather scarce [1]. It can also tolerate low dissolved oxygen levels [2] and during the winter, when water temperature drops down significantly, this gregarious omnivorous fish may bury half way into the pond bottom mud and stay in a dormant sedentary state until spring, the spawning season [3]. This fish is mainly cultivated in the northwestern areas of Spain for its good quality meat as well as for being a valuable recreational ornamental species. Its culture is intensifying across Europe, with translocation of fish and their diseases as a result [4]. Fish can act as definite or intermediate host for parasites. Though most of them behave as saprophytes, these parasites, under certain conditions, may become pathogenic and pose a serious threat to its health status. Thus, any slight, natural or anthropogenic, changes in the rearing conditions can modify the host-parasite relationship leading to outbreaks and high mortalities [5].
The increasing impact of some parasitoses on fish health has enhanced the need for deeper studies on piscine parasite fauna especially when knowledge on fish parasites and their impact on fish are currently very scarce. These deeper studies on the parasite-host-environment system will allow not only a more efficient treatment, but also prevention. This three-way interaction, known as disease epidemiological chain [6], can be observed and analyzed using the valuable tools of the epidemiological investigation [7] to provide useful information on the nature and extent at which certain factors may influence the process of infection. In farmed hosts, like the ones of the present study, culture conditions and management practices are the determinants of epidemiological factors. The bio-geographical location, characterized by abrupt seasonally changes in environmental factors, mainly water temperature, may also play an important role. The present cross-sectional epidemiological study was designed in a tench fish farm located in the Northwestern region of Spain in order to provide the fish farmers with more information on how to deal with some parasitoses (parasitic diseases) that affect their cultured fish. The general objective of such a study was to determine the impact of both the microhabitat (host) and the microhabitat (environment) parameters on the presence of the different taxonomic groups that we found. More specifically, we aimed to establish the possible associations of the factors involved and the infection prevalence/intensity of the parasites, as well as the quantification of the risk or likelihood associated with their presence.
Variable |
Type of variable – value |
General characteristics |
|
Sampling point (municipality) |
Nominal |
Location |
Nominal |
Sampling date |
Date |
Season |
Ordinal |
Physicochemical parameters of the water |
|
Temperature (ᵒC) |
Quantitative |
Dissolved Oxygen (ppm) |
Quantitative |
pH |
Quantitative |
Conductivity (µSiemens/cm) |
Quantitative |
Turbidity (FTU)* |
Quantitative |
Alkalinity (mg/L HCO3-) |
Quantitative |
Hardness (mg/L Calcium and Magnesium CO32- and HCO3-) |
Quantitative |
Ammonium (mg/L NH4+) |
Quantitative |
Nitrites (mg/L NO2-) |
Quantitative |
Nitrates (mg/L NO3-) |
Quantitative |
Total phosphates (mg/L PO4-P) |
Quantitative |
Microbiological parameters of the water |
|
Total Coliforms (CFU/100 mL) |
Quantitative |
Faecal Coliforms (CFU/100 mL) |
Quantitative |
Faecal Streptococci (CFU/100 mL) |
Quantitative |
Sulphite-reducing Clostridia (CFU/20 mL) |
Quantitative |
Total Aerobes at 37ºC (CFU/mL) |
Quantitative |
Total Aerobes at 22ºC (CFU/mL) |
Quantitative |
Appendix 1: Questionnaire 1: Data on the characteristics of the sampling point and the water quality.
Ppm: Parts Per Million; FTU: Formalized Turbidity Unit; HCO3-: Bicarbonates; CO32-: Carbonates; CUF: Colony Forming Units
Variable |
Type of variable – value |
|
|
Characteristics of the fish |
|
|
|
Sex |
Dichotomous |
|
|
Total length (cm)1 |
Continual |
|
|
Weight (g) |
Continual |
|
|
Condition factor (K)2 |
Continual |
|
|
Reproductive status (spawning) |
Dichotomous |
|
|
Parasites |
Infection |
Place (body) |
Intensity |
Ichthyophthirius multifiliis |
Dichotomous |
Nominal |
Ordinal |
Tripartiella sp. |
Dichotomous |
Nominal |
Ordinal |
Gyrodactylus sp. |
Dichotomous |
Nominal |
Ordinal |
Myxobolus sp. (internal organs) |
Dichotomous |
Nominal |
Ordinal |
Appendix 2: Questionnaire 2: Data on the fish and the parasites.
1Total length: length of the fish from the top of the head to the tip of the tail fin.
2Condition factor (somatic condition or Fulton index), according to Fulton equation: K = 100 x weight/lenght3
Data analysis
Once the database was created and filled in with all the required information, a global spreadsheet was generated with all the variables (Appendices 1 & 2) in order to proceed to its statistical and epidemiological analysis, choosing the right tests according to the variable type. In order to process the variables used in the questionnaires with the software SPSS 15.0 for Windows (Chicago, USA), they were given a code number. Aunivariate description of all the variables liableto indicate a variation in the environment or the studied population was performed through the calculation of means and standard deviations, or relative frequencies (percentages) and their 95% confidence intervals, using the software WinEpi [8]. The appropriate null hypotheses were then formulated; in all cases, they were rejected when p-values were lower than 0.050. Depending on the type of variable, the following non-parametric tests used were: Kruskal-Wallis, Mann-Whitney and Chi-square (χ2) or Likelihood Ratio (LR). With them, we could assess any association of host and environmental factors with parasite infection and also decide if the prevalence of exposed and non-exposed to a certain factor was significantly different.
The results recorded for both the physicochemical and the microbiological water parameters (Table 1 & 2) measured during the different studied seasons did not show any significant variations depending on the season. With a quick look on these values shown in Table 1 & 2 and the recommended values for cyprinid farming shown in Appendix 3, one can see that temperature, DO, NO2– and NH4+ values fell within the recommended range, while the pH values were always above the maximum level which may have a negative influence on the health status of the fish.
Water quality parameter |
Standard value for Cyprinids |
Temperature |
Maximum: 28ºC (= maximum temperature allowed at the limit of the mixing zone for more than 98% of the time) |
pH |
Range: 6-9 |
Nitrites |
Maximum: 0.03 mg/L NO2- (reference value) |
Total Ammonium |
Maximum: 0.2 mg/L NH4+ (reference value) |
Dissolved Oxygen (DO) |
Minimum (50% of the water samples): 8 mg/L O2 |
Appendix 3: Parameters of water quality for cyprinids, according to the Freshwater Fish Directive of the European Economic Community, EEC, 1978.
NO2-: Nitrites (mg/L); NH3: Total Ammonium (mg/L); DO: Dissolved Oxygen (mg/L)
|
Spring (N=1) |
Summer (N=2) |
Autumn (N=2) |
Pkw |
Total (N=5) |
T |
19.00 |
21.25 ± 5.30 |
7.10 ± 1.27 |
0.223 |
15.14 ± 7.88 |
DO |
11.80 |
8.55 ± 0.77 |
14.05 ± 1.06 |
0.165 |
11.4 ± 2.84 |
pH |
10.37 |
9.55 ± 0.13 |
9.31 ± 1.23 |
0.368 |
9.62 ± 0.76 |
Cond |
167.00 |
206.50 ± 23.34 |
295.00 ± 55.15 |
0.165 |
234.00 ± 65.25 |
Turb |
4.56 |
4.07 ± 3.06 |
7.25 ± 3.85 |
0.819 |
5.44 ± 2.97 |
Alk |
85.40 |
109.80 ± 17.25 |
164.70 ± 77.64 |
0.301 |
126.88 ± 53.60 |
Hard |
72.00 |
99.00 ± 12.73 |
162.00 ± 50.91 |
0.165 |
118.80 ± 48.63 |
NH4+ |
0.050 |
0.125 ± 0.106 |
0.050 ± 0.000 |
0.472 |
0.08 ± 0.067 |
NO2- |
0.020 |
0.020 ± 0.014 |
0.015 ± 0.007 |
0.823 |
0.018 ± 0.008 |
NO3– |
2.00 |
3.50 ± 2.12 |
2.00 ± 0.00 |
0.472 |
2.60 ± 1.34 |
PO4 |
0.80 |
0.55 ± 0,35 |
0.45 ± 0.35 |
0.398 |
0.56 ± 0.29 |
Table 1: Physicochemical water quality at the fish farm by season.
pKW: p-Values According to Kruskal-Wallis test; T: Temperature (ᵒC); DO: Dissolved Oxygen (ppm); pH; Cond: Conductivity (µS.cm-1); Turb: Turbidity (FTU); Alk: Alkalinity (mg/L); Hard: Hardness (mg/L); NH4+: Ammonium (mg/L); NO2-: Nitrites (mg/L); NO3–: Nitrates (mg/L); PO4: Phosphates (mg/L)
|
Spring (N=1) |
Summer (N=2) |
Autumn (N=2) |
Pkw |
Total (N=5) |
TA 22ºC |
484.0 |
257.5 ± 292.0 |
533.0 ± 202.2 |
0.497 |
413.0 ± 228.3 |
TA 37ºC |
9.0 |
187.0 ± 97.6 |
25.5 ± 6.4 |
0.165 |
86.8 ± 104.0 |
TC |
6.0 |
101.0 ± 140.0 |
5.0 ± 0.0 |
0.729 |
43.6 ± 87.4 |
FC |
0.0 |
80.0 ± 113.1 |
2.0 ± 2.8 |
0.687 |
32.8 ± 71.1 |
FS |
9.0 |
101.0 ± 125.9 |
38.5 ± 30.4 |
0.368 |
57.6 ± 76.9 |
C |
0.0 |
0.0 ± 0.0 |
0.0 ± 0.7 |
0.472 |
0.2 ± 0.5 |
Table 2: Microbiological water quality at the fish farm by season.
pKW: p-Values According to Kruskal-Wallis test; TA 22ᵒC: Total Aerobes at 22ᵒC (CFU/100 mL); TA 37ᵒC: Total Aerobes at 37ᵒC (CFU/100 mL); TC: Total Coliforms (CFU/100 mL); FC: Faecal Coliforms (CFU/100 mL); FS: Faecal Streptococci (CFU/100 mL); C: Clostridia (CFU/20 mL)
From Table 3, one can see that the spawning season for this species was during the spring, when significantly the biggest and the heaviest fish were captured, measured and analyzed.
Fish – tench |
n |
Total Length (cm) |
Weight (g) |
Spawning |
10 |
20.70 ± 6.37 |
141,3 ± 98,21 |
Non-Spawning |
104 |
12.25 ± 2.80 |
27,02 ± 15,27 |
pMW |
|
<0.001 |
<0.001 |
Spring |
10 |
20.70 ± 6.37 |
141.30 ± 98.21 |
Summer |
55 |
11.70 ± 2.86 |
26.55 ± 13.64 |
Autumn |
49 |
12.87 ± 2.63 |
27.55 ± 17.04 |
pKW |
|
<0.001 |
<0.001 |
Table 3: Biometric parameters of the fish by reproductive state (spawning) and season.
pMW: p-Values According to Mann-Whitney test; pKW: p-Values According to Kruskal-Wallis test
In total, four taxonomic groups were identified: two ciliate protozoa (Ichthyophthirius multifiliis and Tripartiella sp.), one myxozoan (Myxobolus sp.) and one monogenean (Gyrodactylus sp.). The results significantly showed that the most prevalent within the fish population were the ciliates, with 100% of it infected by trichodinids and 83.67% infected by I.multifiliis; only 5.56% was infected by the myxozoan and 2.63% by the monogenean. The intensities were only high in the case of Tripartiella sp. (+3 in 82.6% of the fish) and I.multifiliis (+2 in 82.2% of them). In the present study, no mortality was registered and the only symptom observed on the fish was an abundance of white mucus on the gills and body surface of the tenchs especially when the parasite load was high. The analysis also showed statistically significant associations between several factors and parasite abundance, as described below.
Ichthyophthirius multifiliis (“ich”): The trophonts of ich were mainly found on the gills (96.77% of the cases) and skin/gills (3.23%). Some authors [20] state that the development of this phase of the parasite on the gills may occasionally have lethal effects on fish health. In our study, we noticed that the I. multifiliis abundance was significantly associated with season, with the highest intensities appearing in summer. In this season, up to 82.2% of the analyzed fish showed a level +2 (medium), dropping down to 14% in autumn and spring (Figure 2). Our results are in accordance with other authors’ [21,22] who state that Ichthyophthiriasis outbreaks normally occur during the warmer seasons of the year as the water temperature rises. The highest prevalence (%) was recorded in the autumn (83.67% of the fish were parasitized), when temperatures dropped down again (Table 4). According to other authors [23], very high temperatures (>28ᵒC) are lethal for ich, but as they reach their lowest values I. multifiliis seem to “hibernate” on the body of the host. In temperate regions autumn is also a season of abrupt changes, situation which may induce stress and a lower immune response in the fish, hence increasing their susceptibility to parasitization.
|
Spring |
Summer |
Autumn |
Total |
pX2 |
Prevalence (%) |
70.00 |
81.82 |
83.67 |
81.58 |
0.595 |
Table 4: Infection prevalence (%) by I. multifiliis by season.
pX2: p-Value According to Chi-square (χ2) test
Tripartiellasp.: The parasite was only found on the gills of the tenchs (Figure 3). Authors [23-25] mentioned that this is the preferential place for the trichodinids, though they may also be present on the skin of recently hatched larvae [26]. Some intrinsic factors seemed to be linked significantly to the presence of the ciliate. In the first place, the condition factor (K) was significantly lower in the case of the infected fish (Table 5). According to authors [23,27], weakened and stressed fish are the most susceptible to the infection, especially under culturing conditions [17]; though the situation may also be explained the other way around, with a high load of parasites on the fish surface leading to a lower somatic condition. Secondly, the smaller (<13 cm) and younger fish were around 15 times more likely to be infected by the ciliate (Table 6) i.e., both fish size and/or age could act as a risk factor associated with the presence of the parasite. This finding agrees with authors [17,26] who mentioned that the Trichodinasis is more frequent among the larvae and juveniles.
|
Infected Fish (g/cm3) |
Non-Infected Fish (g/cm3) |
pMW |
Condition Factor |
1.47 ± 0.34 |
1.54 ± 1.20 |
<0.001 |
Table 5: Condition factor of the fish infected and non-infected fish by Tripartiella sp.
pMW: p-Value According to Mann-Whitney test
Fish size |
≤ 13 cm |
> 13 cm |
pX2 |
OR |
Prevalence (%) |
84.72 |
26.19 |
0.008 |
15.630 |
Table 6: Size of the fish as risk factor associated with the presence of Tripartiella sp. in the fish farm
pX2: p-Value According to Chi-square (χ2) test; OR: Odds Ratio
Some extrinsic or environmental factors, like season and water quality, showed a significant effect on the presence of the parasite. Table 7 and Figure 4 show that both the prevalence and the intensity of infection reached their highest peaks during the summer season, significantly increasing the likelihood of parasitization by almost 6.5 times (Table 8). This could be attributed to the fish behavior that is conditioned by the temperature fluctuations. This finding agrees with author [28] who mentioned that the multiplication of this parasite may be triggered by a higher activity of the fish. Other authors [5,17,29] related the multiplication of this type of parasite to the more frequent fish contact in shallower water as well as the higher levels of stress during spawning activities which also lead to a weakening in their health status [30,31].
Season |
Spring |
Summer |
Autumn |
Total |
pX2 |
Prevalence (%) |
0.00 |
83.64 |
53.06 |
63.16 |
<0.001 |
Table 7:Infection prevalence (%) by Tripartiella sp. by season.
pX2: p-Value According to Chi-square (χ2) test
Season |
Summer |
Rest of the Seasons |
pX2 |
OR |
Prevalence (%) |
83.64 |
44.07 |
0.001 |
6.487 |
Table 8: Seasonality as risk factor associated with the presence of Tripartiella sp. in the fish farm.
pX2: p-Value According to Chi-square (χ2) Test; OR: Odds Ratio
Finally, according to the results shown in Table 9, some of the studied physicochemical (hardness >100 mg/L, nitrates >3 mg/L & phosphates >0.6 mg/L) and microbiological (total coliforms >100 CFU/100mL, faecal coliforms >0 CFU/100mL, faecal streptococci >50 CFU/100mL & anaerobic clostridia >0 CFU/100mL) water parameters seemed also to be acting as risk factors for the presence of Tripartiella sp. at the fish farm. Other authors [25] point out that poor environmental quality conditions may have an influence on the presence of the parasite. In addition to their negative influence on the fish health, high pH values recorded during the three studied seasons (Table 1) (which have a reflection on the water hardness) may cause an erosion on the fish body surface, with hyper-secretion of mucus as well as cutaneous bleeding and osmoregulation unbalance [32-35], generating chronic-stress [29] that can lead to immunosuppression [23,36] and thus to a higher susceptibility to parasites [37].
|
Exposed |
Non-Exposed |
pX2 |
OR |
|
Hardness >100 mg/L |
Hardness ≤100 mg/L |
|
|
Prevalence (%) |
74.16 |
24.00 |
<0.001 |
9.087 |
|
Nitrates >3 mg/L |
Nitrates ≤3 mg/L |
|
|
Prevalence (%) |
100.00 |
43,24 |
<0.001 |
529.900* |
|
Phosphates >0.6 mg/L |
Phosphates ≤0.6 mg/L |
|
|
Prevalence (%) |
83.54 |
17.14 |
<0.001 |
24.540 |
|
TC >100 CFU/100mL |
TC ≤100 CFU/100mL |
|
|
Prevalence (%) |
100.00 |
43.24 |
<0.001 |
529.900* |
|
FC>0 CFU/100mL |
FC ≤0 CFU/100mL |
|
|
Prevalence (%) |
96.65 |
13.33 |
<0.001 |
143.000 |
|
FS >50 CFU/100mL |
FS ≤50 CFU/100mL |
|
|
Prevalence (%) |
95.65 |
13.33 |
<0.001 |
143.000 |
|
C >0 CFU/20mL |
C ≤0 CFU/20mL |
|
|
Prevalence (%) |
89.66 |
54.12 |
<0.001 |
7.348 |
Table 9: Physicochemical and microbiological water quality as risk factor associated with the presence of Tripartiella sp.
*Null data: Approximate Calculations (WinEpi); pX2: p-Value According to Chi-square (χ2) test; OR: Odds Ratio; TC: Total Coliforms; FC: Faecal Coliforms; FS: Faecal Streptococci; C: Clostridia
Myxobolussp.: The myxozoan was only present in the fish internal organs (liver, kidney and gonads). According to authors [26], some species of the genus Myxobolus seem to be specific to certain organs. It has also been found on the skin, gills and fins [23]. Table 10 indicates that only the females were infected by Myxobolus sp. (prevalence of 5.56%). This may be related to their weakening after the spawning season [30,31]. From Table 11, one can see that the parasite was only present in the summer (prevalence of 5.45%). Some authors [38] stated that some myxosporidia cause epizootic diseases only during the warmer seasons of the year.
Sex |
Male |
Female |
Total |
pLR |
Prevalence (%) |
0.00 |
5.56 |
3.33 |
0.077 |
Table 10: Infection prevalence (%) by Myxobolus sp. by sex.
pLR: p-Value According to Likelihood Ratio
Season |
Spring |
Summer |
Autumn |
Total |
pLR |
Prevalence (%) |
0.00 |
5.45 |
0.00 |
2.63 |
0.108 |
Table 11: Infection prevalence (%) by Myxobolus sp. by season.
pLR: p-Value According to Likelihood Ratio
Gyrodactylus sp.: The metazoan was found on the fish skin (66.67% of the cases) and the gills (33.33%). Though most species seem to be able to move actively around the host surfaces [39,40], where they feed on epithelial cells, mucus and blood [17,41]. However, under situations of intense infection, some species may also be found in the mouth and pharyngeal cavities [42]. In general, 2.63% of the tenchs were infected, reaching 5.45% in the summer (Table 12) probably due to rising temperatures [43-45]. Besides the low prevalence, also intensity was low in 100% of the cases.
Season |
Spring |
Summer |
Autumn |
Total |
pLR |
Prevalence (%) |
0.00 |
5.45 |
0.00 |
2.63 |
0.108 |
Table 12: Infection prevalence (%) by Gyrodactylus sp.by season.
pLR: p-Value According to Likelihood Ratio
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