Diagnosis of bovine gastrointestinal parasites: comparison of different techniques and different solutions

OBJECTIVES : Cattle are important livestock species in Nigeria. But disease agents, especially gastrointestinal (GI) parasites, hinder their productivity. Non availability of rapid and affordable tools for GI parasite diagnosis poses problem to practitioners thus, the use of rapid, highly sensitive diagnostic techniques is required. One hundred faecal samples from cattle were analyzed through Simple Faecal Flotation (SFF), Modied Centrifugal Faecal Flotation (MCFF) and Mini-FLOTAC ® techniques (MFT) using Sodium Chloride, Brine, Sugar, Salt/Sugar and Zinc Sulphate at specic gravity of 1.2, 1.23, 1.25, 1.3 and 1.3 respectively as Flotation Solutions (FS). RESULTS : 100% GI parasite prevalence of was recorded with the following distribution: Strongyle (99%), Strongyloides spp (97%), Neoascaris vitulorum (78%), Trichostrongylus (56%), Nematodirus (46%), Capillaria spp (14%) and Trichuris spp (6%), Moniezia benedini (24%), Moniezia expansa (16%) , Taenia-like egg (3%), Schistosoma eggs (3%) and Eimeria oocyst (100%). Using Salt/Sugar the sensitivity of the techniques was 61.99%, 58.49% and 54.24%. for MFT, SFF and MCFF respectively. Considering the sensitivity of Salt/Sugar FS with availability and affordability its use as a routine FS is advocated. The ease and rapid use of Mini-FLOTAC in low economy country in addition to safety of diagnostic staff and the possibility of re-usage.


Introduction
Livestock contribute to natural, nancial, human, physical and social lives in different ways and to different degrees within small holder dairy, crop-livestock and livestock-dependent systems [1] Cattle are the single most important livestock species in Nigeria and play a signi cant role in the socioeconomic life of the people. In addition to the production of meat and milk, cattle provide draught power for cultivation of the agricultural lands of many peasants. Skins and hides are also important components of the livestock sector in generating foreign export earnings [2] Gastrointestinal helminthes are ubiquitous parasitic agents of livestock especially ruminants and are known to limit cattle production in many climatic areas and countries [3] While most helminth parasites reside in the intestines, they can also be seen in the stomach, bile duct, lungs, liver and even gall bladder of ruminants [4].
There appears to be almost endless possibilities in the number of substances that could be used in the otation technique. Indeed, everything from simple table salt and table sugar (sucrose) to other chemicals have been used in otation procedures. Although solutions with speci c gravities of 1.15 to 1.55 have been investigated, efforts have generally been focused on solutions with speci c gravities between 1.18 and 1.30 [6,7,8,9] As polyparasitism is very common and in response to the recent trend toward integrated control of multiple parasitic diseases, there is a need for sensitive and accurate diagnostic tools that are simple to apply [10]. In this wise, Cringoli et al. [11] developed a multivalent technique, denominated FLOTAC®, for qualitative and quantitative copro-microscopic diagnosis of parasitic infections in animals and humans.
The FLOTAC® technique proved to be more sensitive compared to the McMaster technique in a survey of anthelminthic resistance in cattle [12]. However, FLOTAC® is more time consuming and requires a centrifuge for plates which limits its adoption for use where a centrifuge is not available or electric power supply is irregular, as is the case with several developing countries like Nigeria. To overcome this constraint, a more user-friendly technique, mini-FLOTAC®, was developed by the same researchers in 2013 [13]. Mini-FLOTAC® does not require centrifugation and can detect as low as 10 eggs per gram of faeces.
We are not aware of studies regarding the evaluation of this technique in Nigeria. Hence this study on the

Parasitological techniques
The techniques used were: Simple Faecal Flotation, Modi ed Centrifugal Faecal Flotation and Mini-FLOTAC® [16,17,13] Data Analysis Data obtained were subjected to descriptive statistical analysis using percentages for prevalence rates across different breeds, age, body condition score and sex.
Results from this study were also applied by means of comparisons of positive data from faecal samples obtained by parasitological techniques, to determine, their sensitivity. Total positive from all the three techniques and the otation solutions was taken to be the gold standard [18] to cater for the merits and demerits of each technique and oatation solutions.

Results
Of the one hundred cattle sampled all were positive for at least one gastrointestinal parasite as helminth egg or protozoan oocysts (100%). Eleven genera of gastrointestinal parasites were recovered in this study. They include: seven nematodes-Strongyle (99%), Trichostrongylus (56%), Nematodirus (46%), Neoascaris vitulorum (78%), Strongyloides spp (97%), Capillaria spp (14%) and Trichuris spp (6%)); two cestodes: Moniezia benedini (24%), Moniezia expansa (16%) and Taenia egg (3%)); one trematode: Schistosoma spp(3%) and one protozoa: Eimeria (100%) ( In terms of rapidity of the three techniques, simple otation takes two minutes for processing a sample, twenty minutes required for oating and one minute for observation under the microscope (23 minutes all together). Modi ed Centrifugal Faecal Flotation takes seventeen minutes for processing a sample, minimum of ten minutes for oating of parasite elements and one minute for microscopic examination (28 minutes all together). Mini-FLOTAC technique takes three minutes to process a sample, ten minutes for parasite elements to oat and one minute for microscopic examination of the reading disk (14 minutes all together).

Discussion
The ndings of this study showed that all the cattle screened had helminth infection, thus providing valuable information on the burden of helminths among cattle in sampled areas.
The overall prevalence of 100% of GI parasite infection obtained in this study was higher than those by [19] and [20] who reported a prevalence of 47.4% and 41.6% in South-South and South-West Nigeria respectively. It was also higher than 50.8% and 62.1% earlier reported in South-Eastern and South-South Nigeria by [4] and [21] respectively.
The difference in the prevalence obtained could be attributed to the existence of favourable environmental factors necessary for the prolonged survival and development of infective larval stage of most helminths [22], variation in sample size and management system. [23] reported that animals that are solely graze on pasture throughout the year are prone to the effect of seasonal variation of availability of forgeable feed and then difference in plane of nutrition.
Strongyle eggs, Trichostrongylus, Nematodirus, Neoascaris vitulorum, Strongyloides spp, Capillaria spp and Trichuris spp, Moniezia benedini, Moniezia expansa, Taenia egg, Schistosoma eggs and Eimeria oocyst were identi ed in this study using simple otation, modi ed centrifugal faecal otation and Mini-FLOTAC techniques An important observation from this study is that the Capillaria eggs detected is from Muturu breed of cattle sampled in a rural Area. In this area, many people defecate on the farmland and these animals are grazed on the same farmland that had been contaminated with human faeces. Although bovine coprological examination carried out by various researchers in other countries reported Capillaria spp as one of the parasite elements detected [24], to date no such report has been made from cattle in Nigeria.
The sensitivity of Mini-FLOTAC using salt/sugar solution is higher compared to the salt/sugar modi ed centrifugal faecal otation. This diagnostic assessment clearly indicated that the solution was the best under the Mini-FLOTAC technique.
In general, diagnostic assessment of the ve otation solutions clearly indicated that salt/sugar solution gave the optimum results under each faecal test for the three techniques compared to other solutions suggesting that the solution is highly e cient in the diagnosis of parasite elements, Salt/sugar is easy to prepare, the solute is readily available, does not grow mouldy unlike sugar solution, does not crystalize or form cast, does not require preservative, it is not sticky or messy to work with, non-toxic to the environment and nally it has excellent clarity under the microscope.
One plausible explanation for the superior performance of the salt/sugar solution under each technique is that salt helps in homogenization of faeces and sugar which is more viscous make it the Parasite elements appear clearer with increasing chance of detection [25].

For the diagnostic techniques, Mini-FLOTAC produced optimum results compared to the other two conventional techniques (Simple Faecal Flotation and Modi ed Centrifugal Faecal Flotation) especially
with the use of salt/sugar as otation solution. Also, Mini-FLOTAC apparatus is heat resistant, re-useable after thorough washing, has excellent clarity under the microscope, does not require centrifugation and is good for both qualitative and quantitative faecal analysis.
Findings from this study showed that only salt/sugar solution was highly e cient in detecting the eleven genera of bovine gastrointestinal parasites identi ed in this study using Mini-FLOTAC technique. This further support RCV/FAO recommendation that salt/sugar otation solution is a general-purpose otation solution.
This is the rst-time salt/sugar and sugar otation solutions were used with Mini-FLOTAC technique, just recently Salt/Glucose was used by the inventors [13] on a test running scheme, salt solution (Nacl) and zinc sulphate are the only two otation solutions used for the Mini-FLOTAC technique [26] The excellent performance of salt/sugar solution under Mini-FLOTAC technique from this study supported the report by [26] that the sensitivity of the Mini-FLOTAC technique is highly dependent on the otation solution used.
In terms of diagnosis of co-infection, Mini-FLOTAC technique performed better than the other two techniques showing that Mini-FLOTAC is a more sensitive technique.
The only protozoa parasite detected by the three parasitological techniques is Eimeria oocyst which shows that it is the most prevalence protozoa parasite in the sampled area. This nding is like that of [13] which reported Eimeria oocyst as the only protozoan parasite detected using Mini-FLOTAC technique.
Based on the performance of salt/sugar otation solution under each technique for this study, the use of salt/sugar as otation solution in the laboratory is recommended.
For routine diagnosis, Mini-FLOTAC can be adopted because of the ease of usage.
The use of Mini-FLOTAC needs to be promoted in developing countries particularly in the laboratory with resource-limited settings.

LIMITATIONS
Mini-FLOTAC to work with is quite expensive for routine use in Nigeria Paucity of funds to work with to get quality work done.