Introduction
India is the largest producer of milk in the world and produced around 194,800 thousand tonnes of milk, that accounted for 40.41% of the world's production of milk in 2020.1 India could achieve this feat due to its cattle population (305,500 thousand heads), which is the largest in the world. 2 Along with the numerous benefits which is brought about by this huge cattle population, there also arises a risk of numerous zoonotic infections which can adversely affect the cattle as well as their handlers. Amongst the major infections that can affect the cattle are Intestinal worm infestations.
Intestinal worm infestations are amongst a major constraint in running a profitable dairy industry. Intestinal worm infestations are more common in tropical and sub-tropical countries which includes India. 3 Gastrointestinal (GI) parasites can cause considerable global and domestic economic losses as a consequence of impaired weight gain, digestive tract disturbances, decreased production, impaired reproductive performance, condemnation of affected organs and mortality in infected animals. 4 To counter this menace we need to plan and implement effective deworming programs which can be planned only after having a proper base line data and handy techniques for detection of the parasites which can be implemented in the field. Many if the cattle parasites are also transmission to man.
The current study was done with the objective of determining the prevalence of Intestinal worm infestation amongst the cattle population in a village of the sunderban area of West Bengal and to determine the utility of a novel solution (454 gm of Sucrose to 300 ml of very hot distilled water, along with 45 ml 95% ethanol) vis-à-vis standard salt solution.
Materials and Methods
Ethical approval
Ethical approval from the institutional ethical committee was taken before commencement of the study. Permission was also duly taken from Krishi Vikas Kendra, Nimpith, South 24 Parganas as the village Kaikhali, in which the study was being conducted came under their ambit. Owner of each cattle head was properly explained about the procedure being conducted and only after due consent, dung sample was collected. The results of the test were conveyed to the cattle owners as well as the Krishi Vikas Kendra, Nimpith.
Study area
The study was conducted in Kaikhali village, Nimpith. It was under the jurisdiction of South 24 Parganas district of West Bengal. The village lies adjacent to the Matla River and is geographically located at the peripheral area of sunderbans. The area receives plenty of rainfall and has typical tropical climate.
Study population
Sample size calculation for number of cattle heads was calculated using the Cochrane formula,
Cattle with acute illnesses, pregnant cattle and those which were untagged were left out of study. Cattle were selected randomly from households as well as small farms (maximum herd size 8).
Study technique
Fresh faecal sample was collected directly from the rectum of the cattle after properly restraining the animal. Around 20 grams of faecal sample was collected from each cattle head. All aseptic precautions like usage of gloves and sterile containers were done for the procedure. The study was conducted in the month of August 2022. The sample were transported back to the laboratory and examined on the very same day of collection. The sample was processed using two types of floatation technique, one using the conventional Salt floatation and other using a Novel Solution (454 gm of Sucrose to 300 ml of very hot distilled water, along with 45 ml 95% ethanol). If any of the two techniques yielded a parasite ova or egg, it was considered positive.
Results
Amongst the 50 heads of cattle studied, majority were females (82%) and >2 years of age (76%). Most cattle were of native varieties (76%) and only a minority were cross breeds (24%), no pure foreign breed cattle were observed. The cattle were mostly owned by Individual households (60%) and two farms in the region owned the rest (40%) of the cattle heads. Of all the cattle examined, 29 specimen were found to be positive (As per the working definition in the study, dung sample which showed parasitic specimen in either of the floatation technique were counted as positive).
Table 1
Distribution of Cattle according to the their Intestinal Parasitic Prevalence and associated variables.
Along with cattle dung, 8 number of soil specimen were collected from random areas in the village, near the vicinity of the cattle sheds. The soil specimens too were checked for the presence of parasitic specimen. Of the 8 specimens examined, none showed any parasitic presence in Salt Floatation while 3 showed the presence of Strongyloides spp. in floatation done through Novel Solution.
On further analysis through Regression Analysis, we found that there is no significant association between presence of Intestinal Parasitic infestation and any of the other variables.
Table 2
Regression analysis of Presence of Intestinal parasitic infestation along with other variables.
Of the 50 total samples observed, we found out that 29 samples (58%) are positive for intestinal worm infestation i.e., in at least one of the two floatation technique we could identify a parasitic specimen. The occurrence rate of Individual species was – Strongyloides spp. -16 (32%), Balantidium coli- 12 (24%), Entamoeba spp. – 2 (4%) and Amphistome – 1 (2%). Upon conducting microscopy after floatation with Standard Salt Solution we found that the prevalence as:- Strongyloides spp. - 8 (16%), Balantidium coli- 6 (12%), Entamoeba Sp. – 1 (2%) and Amphistome – 1 (2%). In contrast to it, more isolates could be identified through the Novel solution, Strongyloides - 10 (20%), Balantidium Coli- 9 (18%), Entamoeba Sp. – 1 (2%) and Amphistome – 0 (0%). The description of solution wise isolate is further elaborated in Table 1.
Table 3
Flotation Solution wise species isolated
|
Parasitic Species |
Salt Floatation |
Novel Solution Floatation |
|
Strongyloides spp. |
8 |
10 |
|
Balantidium coli |
6 |
9 |
|
Entamoeba Sp. |
1 |
1 |
|
Amphistome spp. |
1 |
0 |
|
Total Positive |
16 |
20 |
There was a difference observed upon comparison of positivity rate between the two floatation techniques. Floatation done through Novel Solution showed a higher yield as compared to standard Salt floatation technique. On further analysis to check for the agreement between the two techniques, we found out that Cohen’s k is 0.0517 which indicates moderate agreement.
New sugar alcohol mixture was found to be very good for parasite-infested samples containing Balantidium coli, Hookworm and others.
Discussion
Most commonly collected samples for studying parasites spreading from cattle to man, are faecal and soil samples. So far there are very few solutions which show positive results in both faecal and soil samples. In this study we collected dung samples of cattle from the rectum and also soil samples around the cattle holding area. Concentration methods like floatation and sedimentation are necessary for detection of ova and cysts in stool samples because of poor sensitivity of direct microscopy of stool samples. Proper concentration technique is further necessitated due to intermittent shedding of parasitic cysts and ova in stool. 5 There are many standard solutions and methods used in concentration technique for the detection of parasitic ova from the stool samples, like Saturated saline and Formol-ether sedimentation. 6 A major disadvantage of standard floatation methods like Saturated Saline floatation is that cyst and ova walls often collapse. 7 This was not observed in samples processed through our novel solution.
Faeces of domestic animals should be disposed properly due to the risk of transmission of parasites like Cryptosporidium spp., soil transmitted helminths and others. 8 However, as most of the domestic animal owners do not take proper precautions during collection and disposal of dung, there may be chances of transmission of these parasites directly and also indirectly through soil, causing infestation in humans. Dog faeces can transmit many parasitic infections to man, like roundworms and thread worms. 9 Studies have found high prevalence of parasites like coccidian cysts and Trichostrongylus spp. in cow dung. 10 Researchers from Ghana have also found high burden of roundworms like Ascaris spp. in faecal matter of cattle which can infect man, especially children. 11 A study from Kolkata, India had also found high burden of protozoa like Entamoeba histolytica in cow dung. 12 So both dung and soil samples were examined for parasitic ova and simultaneous comparison was made between our sugar-ethanol and saturated salt solution. Our results have shown positive results in both methods. There are many concentration methods, each with their own advantages and disadvantages. For example, formol-ether sedimentation method is better for recovery of Schistosoma eggs. 13 The disadvantage of saturated salt solution floatation is that delay in examination can lead to distortion. 14 However floatation methods are overall better than sedimentation due to formation of a cleaner material for examination. 15
New floatation and concentration methods which preserve all ova and cysts equally well, are the need of the hour for stool samples. Our new method was very effective and gave good results for hookworm eggs. Compared to other standard solutions it is economical and can also be prepared easily. Results were good and reproducible for Balantidium coli, Hookworm and other parasites with the novel solution. Thus, it can be recommended to be used in human stool samples also since many of these parasites can cause both human and animal infections. The novel preparation is cheap and effective and sometimes more sensitive as compared to Saturated saline. As far as we know, nobody has tried this method for concentration method for parasitic ova and yeasts. Sucrose will help in maintaining hyperosmolarity facilitating floatation and alcohol will help in preservation. So, these findings are important from microbiological angle, public health viewpoint as well as from one health and zoonotic perspectives.
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