CN114381375A - Treatment method and application of chicken Eimeria maxima excrement mucus - Google Patents
Treatment method and application of chicken Eimeria maxima excrement mucus Download PDFInfo
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Abstract
The invention belongs to the field of animal waste treatment, and discloses a treatment method and application of chicken Eimeria maxima waste mucus, wherein the treatment method comprises the following steps: mixing glutaraldehyde and mucus-containing chicken Eimeria maxima manure liquid, sterilizing, adding phosphoric acid to adjust pH to 1.5-2.5, and adding pepsin to decompose mucus in the manure liquid to obtain turbid liquid; and (4) treating the turbid liquid by ultrasonic cavitation to obtain the fecal treatment liquid. The method comprises the steps of killing bacteria in the liquid dung by glutaraldehyde, decomposing mucus of the liquid dung in the liquid dung by pepsin to reduce the adhesiveness of the mucus, and finally releasing chicken Eimeria maxima oocysts in the liquid dung by ultrasonic cavitation treatment.
Description
Technical Field
The invention belongs to the field of animal waste treatment, and particularly relates to a treatment method and application of chicken Eimeria maxima waste mucus.
Background
In recent years, the use of anticoccidial drugs has been prohibited or restricted due to drug resistance of coccidian drugs and the increasing importance of food safety in the country. Therefore, more and more breeding enterprises use coccidian vaccines to prevent and control chicken coccidiosis. One of the coccidia vaccines, eimeria maxima, is predominantly parasitic in the middle small intestine, from below the duodenum until the vitelline pedicle, and in severe infections, the lesions spread throughout the small intestine. The small intestine is flaccid and filled with liquid, yellow or orange mucus is usually contained in the intestinal cavity, epidemic diseases which are characterized by enteritis, diarrhea, growth retardation, reduced feed conversion rate and even death are caused, and great economic loss is caused to the breeding industry. The batch preparation of the chicken coccidian vaccine needs to separate coccidian oocysts from excrement. The feces discharged by the infected Eimeria maxima contain a large amount of mucus (shown in figure 1), and the mucus contains a large amount of Eimeria maxima oocysts (shown in figure 2), so that the mucus has strong adhesiveness and the oocysts are not easy to release and obtain. In mass production, the excrement can be stored in a low-temperature environment without being immediately treated, but the excrement is very suitable for the propagation of bacteria due to a large amount of intestinal mucus, undigested feed and other substances. The fermentation expansion of the feces occurs during the propagation of the bacteria (see fig. 17), and the handling capacity of the feces is increased virtually. The existing coccidian oocyst sorting technology comprises the following steps: the mucilage is rubbed and ground mechanically or manually. Mechanical grinding damages oocysts, affects sporulation and development synchronicity and sporulation rate of giant oocysts, and is not favorable for vaccine quality. Mucus in the excrement is manually rubbed by hands, so that the recovery rate of oocysts is low due to insufficient release, the separation efficiency is reduced, and the separation time is prolonged. Improvements are needed that would not otherwise be satisfactory for large-scale production of coccidial vaccines. And the mucus of the excrement is not treated by the existing coccidian oocyst separation technology.
Therefore, the problem of processing oocysts of the mucus-containing feces due to enteritis caused by infection of the oocysts of the Eimeria maxima, falling off of intestinal mucosa and discharge of a large amount of blood mucus with relatively strong viscosity in the feces is solved.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a treatment method and application of chicken Eimeria maxima stool mucus, and the treatment method improves the separation efficiency and oocyst recovery rate of Eimeria maxima oocysts on the premise of not damaging oocysts, and the oocyst recovery rate is improved to more than 95% from the original 70%.
The invention conception is as follows: the invention provides a method for treating chicken Eimeria maxima excrement mucus, which comprises the steps of treating excrement liquid by combining glutaraldehyde and pepsin, and then completely dispersing and releasing oocysts on the premise of not damaging the oocysts after ultrasonic cavitation treatment, so that the separation efficiency and the recovery rate of the Eimeria maxima oocysts are improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for treating chicken Eimeria maxima excrement mucus comprises the following steps:
(1) mixing glutaraldehyde and chicken Eimeria maxima manure liquid containing mucus, sterilizing, adding phosphoric acid to adjust pH to 1.5-2.5, and adding pepsin to decompose mucus in the manure liquid to obtain turbid liquid;
(2) and processing the turbid liquid by ultrasonic cavitation to obtain the fecal treatment liquid.
Preferably, the step (2) further comprises sieving the fecal treatment fluid, collecting the filtrate, centrifuging the filtrate, and collecting the precipitate obtained by centrifugation; adding saturated saline solution into the precipitate, stirring, centrifuging, floating, collecting supernatant, adding water, centrifuging, and collecting precipitate to obtain chicken Eimeria maxima oocysts.
Further preferably, the number of said sieving is 1 to 5.
Further preferably, the screening is to mix the fecal treatment fluid and water according to the volume ratio of 1 (1-2) and then pass through a 200-mesh and 500-mesh screen.
Further preferably, the rotating speed of the centrifugation is 2000-3000r/min, and the time of the centrifugation is 1-5 min.
More preferably, the liquid-solid ratio of the saturated saline to the precipitate is (1-3): 1L/kg.
Further preferably, the volume ratio of the supernatant to the water is 1 (5-10).
Preferably, in step (1), the liquid-solid ratio of glutaraldehyde and pepsin is (8-16): (5-10) ml/g.
Preferably, in the step (1), the liquid dung is prepared by mixing the chicken Eimeria maxima excrement containing mucus and water according to the solid-liquid ratio of (1-3): (0.9-0.93) g/mL.
Glutaraldehyde: has effects in killing bacteria propagules, inhibiting bacteria growth, and preventing feces from swelling due to fermentation. If other general bactericides such as iodine preparations are used and stored for 4 hours at a temperature of 30 ℃, the liquid manure generates a large amount of acid and gas, and thus mold growth occurs.
Pepsin: the preparation has the functions of decomposing intestinal mucosa tissue shedding caused by enteritis and discharging blood mucus with stronger viscosity from excrement so as to reduce the viscosity of mucus, and has the functions of sterilizing and reducing the viscosity of mucus on chicken Eimeria maxima excrement by combining with glutaraldehyde so as to release part of chicken Eimeria maxima oocysts from the excrement, and then the mucus is uniformly dispersed and crushed by ultrasonic cavitation treatment so as to completely release the chicken Eimeria maxima oocysts in the excrement.
Preferably, in the step (2), the specific operation process of the ultrasonic cavitation treatment is as follows: firstly, placing a layer of 800-plus-1200-mesh nylon filter screen on the inner wall of an ultrasonic treatment machine, then adding 1000-plus-1100 g of steel balls into the nylon filter screen, finally adding the turbid liquid into the nylon filter screen, and carrying out ultrasonic treatment for 10-30 minutes under the ultrasonic power of 300-600W and the frequency of 20-40 KHz.
The 800-1200 mesh nylon filter screen is used because the aperture of the 800-mesh nylon filter screen is 18 microns, the aperture of the 1200-mesh nylon filter screen is 12 microns, and the size of the giant oocysts is 30.7 multiplied by 21.0 microns, so that the separated oocysts can be isolated by 100 percent and enter the ultrasonic machine, and other smaller substances after treatment can pass through the nylon filter screen.
More preferably, the aperture of the nylon filter screen is 1000 meshes.
Principle of ultrasonic cavitation treatment: when ultrasonic waves are spread along gaps of liquid in turbid liquid, molecules of the liquid are transmitted by the energy of the ultrasonic waves and have energy, the molecules interact with each other to generate a large number of bubbles, the bubbles form a precondition for cavitation, and the bubbles are broken to generate huge energy to break the whole liquid when the energy is gathered to a certain degree.
The invention utilizes the cavitation effect of ultrasonic cleaning, namely countless bubbles in the turbid liquid are quickly formed and quickly implode, and the mucus in the turbid liquid is dissolved by the impact generated by the cavitation effect and the giant coccidian oocysts in the turbid liquid are released.
The viscosity of mucus in the excrement of the chicken Eimeria maxima is reduced, firstly, the mucus in the excrement is continuously rubbed with steel balls under the action of pepsin and secondly, the mucus in the excrement is continuously rubbed with the steel balls under the action of ultrasonic waves, so that the mucus tissue is decomposed, the adhesion capacity of the mucus is reduced to the maximum extent, the chicken Eimeria maxima oocysts can be quickly separated, the separation efficiency is improved, and no adverse effect or damage to the oocysts can be obtained under the condition that the coccidian oocysts are sporulated.
The invention also provides the application of the treatment method in the separation of the animal parasite oocysts.
Advantageous effects
(1) The method comprises the steps of killing bacteria in the liquid dung by glutaraldehyde, decomposing mucus in the liquid dung by pepsin to reduce the adhesiveness of the mucus, and finally processing the mucus in the fine crushed liquid dung by ultrasonic cavitation to obtain the coccidian oocysts, wherein the separation efficiency of the coccidian oocysts is improved by more than 25.0%. Ultrasonic cavitation is carried out on the liquid dung, ultrasonic waves can penetrate through the solid to enable the whole liquid medium to vibrate and generate cavitation bubbles, so that mucus in excrement and the steel balls are continuously rubbed under the action of the ultrasonic waves only when the liquid dung is soaked in water, the tissue of the mucus in the excrement is decomposed, the adhesion capacity of the mucus is reduced to the maximum extent, the chicken Eimeria maxima oocysts are rapidly separated, and the recovery rate of the coccidium oocysts is improved to be more than 95% from the original 70%.
(2) The invention has simple operation and obvious effect, and has no adverse effect and damage to the oocysts. A layer of 800-1200-mesh nylon filter screen is placed on the inner wall of the ultrasonic treatment machine, so that not only can 100% of separated oocysts be isolated from entering the ultrasonic treatment machine, but also other smaller substances after treatment can pass through the nylon filter screen.
Drawings
FIG. 1 is a mucous membrane of the feces of giant insect;
FIG. 2 is a diagram of giant insect feces with large amount of oocysts under mucoscopy;
FIG. 3 is a microscopic examination of the residue of oocysts from feces obtained in example 1 of the present invention;
FIG. 4 is a diagram showing the residual status of oocysts of fecal debris according to example 2 of the present invention;
FIG. 5 is a graph showing residual fecal debris oocysts of comparative example 1 of the present invention;
FIG. 6 is a graph showing residual oocysts of fecal debris according to comparative example 2 of the present invention;
FIG. 7 is a graph showing the concentration at the 5 th filtration of comparative example 3 according to the present invention;
FIG. 8 is a graph showing the concentration at the 6 th filtration of comparative example 3 according to the present invention;
FIG. 9 is a microscopic examination of fecal dregs of comparative example 3 according to the present invention;
FIG. 10 is a microscopic examination of the precipitate of comparative example 3 according to the present invention;
FIG. 11 is a graph showing the fecal sludge filtration of comparative example 1 of the present invention;
FIG. 12 is a graph showing the fecal sludge filtration of comparative example 2 of the present invention;
FIG. 13 is a graph showing the fecal sludge filtration of comparative example 3 of the present invention;
FIG. 14 is a graph showing the fecal sludge filtration of comparative example 4 of the present invention;
FIG. 15 is a microscopic examination of fecal dregs of comparative example 5 of the present invention;
FIG. 16 is a microscopic examination of fecal dregs of comparative example 6 according to the present invention;
FIG. 17 is a graph showing the fermentation and swelling of feces;
FIG. 18 is a diagram showing sporulation of E.maxima according to example 1 of the present invention.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.
The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.
Example 1
The method for treating chicken Eimeria maxima fecal mucus comprises the following steps:
(1) mixing 390kg of chicken Eimeria maxima excrement containing mucus with 130L of water to obtain excrement liquid, adding 8L of glutaraldehyde for mixing, sterilizing, adding phosphoric acid to adjust the pH value to 2.0, adding 5kg of pepsin for mixing, and decomposing the mucus in the excrement liquid to obtain turbid liquid;
(2) firstly, placing a layer of 1000-mesh nylon filter screen on the inner wall of an ultrasonic processor, then adding 1000g of steel balls into the nylon filter screen, finally adding the turbid liquid into the nylon filter screen, and carrying out ultrasonic cavitation on the finely-divided viscous liquid for 20 minutes under the ultrasonic power of 600W and the frequency of 20-40KHz to obtain a fecal treatment liquid;
(3) sieving the fecal treatment liquid and water with a 500-mesh sieve according to a volume ratio of 1:1 to obtain fecal residues and filtrate, collecting the filtrate, centrifuging the filtrate at a rotation speed of 2000r/min for 2 minutes, removing supernatant, and collecting precipitate;
(4) adding saturated salt water solution into the precipitate according to the liquid-solid ratio of 2:1, stirring, centrifuging at the rotating speed of 3000r/min for 3 minutes, collecting the floating liquid (supernatant), adding water into the floating liquid according to the volume ratio of 5:1, centrifuging at the rotating speed of 2500r/min for 3 minutes, and collecting the centrifugal precipitate to obtain the coccidian oocysts.
The coccidian oocysts obtained in example 1 were counted.
FIG. 18 is a diagram showing the sporulation of E.maxima according to example 1 of the present invention, and it can be seen from FIG. 18 that the ultrasonic cavitation treatment did not adversely affect and damage oocysts.
Example 2
The method for treating chicken Eimeria maxima excrement mucus comprises the following steps:
(1) mixing 33kg of chicken Eimeria maxima excrement containing mucus with 15L of water to obtain an excrement liquid, adding 1L of glutaraldehyde for mixing, sterilizing, adding phosphoric acid for adjusting the pH value to 2.0, adding 0.5kg of pepsin for mixing, and decomposing the mucus in the excrement liquid to obtain a turbid liquid;
(2) firstly, placing a layer of 1000-mesh nylon filter screen on the inner wall of an ultrasonic processor, then adding 1000g of steel balls into the nylon filter screen, finally adding the turbid liquid into the nylon filter screen, and carrying out ultrasonic cavitation on the finely-divided viscous liquid for 20 minutes under the ultrasonic power of 600W and the frequency of 20-40KHz to obtain a fecal treatment liquid;
(3) sieving the fecal treatment liquid and water with a 500-mesh sieve according to a volume ratio of 1:1 to obtain fecal residues and filtrate, collecting the filtrate, centrifuging the filtrate at a rotation speed of 2000r/min for 2 minutes, removing supernatant, and collecting precipitate;
(4) adding saturated salt water solution into the precipitate according to the liquid-solid ratio of 2:1, stirring, centrifuging at the rotating speed of 3000r/min for 3 minutes, collecting the floating liquid (supernatant), adding water into the floating liquid according to the volume ratio of 5:1, centrifuging at the rotating speed of 2500r/min for 3 minutes, and collecting the centrifugal precipitate to obtain the coccidian oocysts.
The coccidian oocysts obtained in example 2 were counted.
Example 3
The method for treating chicken Eimeria maxima excrement mucus comprises the following steps:
(1) mixing 264kg of chicken Eimeria maxima excrement containing mucus with 160L of water to obtain an excrement liquid, adding 11L of glutaraldehyde for mixing, sterilizing, then adding 264g of phosphoric acid for adjusting the pH value to 2.0, then adding 5kg of pepsin for mixing, and decomposing the mucus in the excrement liquid to obtain a turbid liquid;
(2) firstly, placing a layer of 1000-mesh nylon filter screen on the inner wall of an ultrasonic processor, then adding 1000g of steel balls into the nylon filter screen, finally adding the turbid liquid into the nylon filter screen, and carrying out ultrasonic cavitation on the finely-divided viscous liquid for 20 minutes under the ultrasonic power of 600W and the frequency of 20-40KHz to obtain a fecal treatment liquid;
(3) sieving the fecal treatment liquid and water with a 500-mesh sieve according to a volume ratio of 1:1.5 to obtain fecal residues and filtrate, collecting the filtrate, centrifuging the filtrate at a rotation speed of 2000r/min for 2 minutes, removing supernatant, and collecting precipitate;
(4) adding saturated salt water solution into the precipitate according to the liquid-solid ratio of 2:1, stirring, centrifuging at the rotating speed of 3000r/min for 3 minutes, collecting the floating liquid (supernatant), adding water into the floating liquid according to the volume ratio of 5:1, centrifuging at the rotating speed of 2500r/min for 3 minutes, and collecting the centrifugal precipitate to obtain the coccidian oocysts.
The coccidian oocysts obtained in example 3 were counted.
Example 4
The method for treating chicken Eimeria maxima fecal mucus comprises the following steps:
(1) 610.5kg of chicken Eimeria maxima excrement containing mucus is mixed with 130L of water to obtain liquid dung, 11L of glutaraldehyde is added for mixing, sterilization is carried out, 611g of phosphoric acid is added for adjusting the pH value to 2.0, 7.5kg of pepsin is added for mixing, and the mucus in the liquid dung is decomposed to obtain turbid liquid;
(2) firstly, placing a layer of 1000-mesh nylon filter screen on the inner wall of an ultrasonic processor, then adding 1000g of steel balls into the nylon filter screen, finally adding the turbid liquid into the nylon filter screen, and carrying out ultrasonic cavitation on the finely-divided viscous liquid for 20 minutes under the ultrasonic power of 600W and the frequency of 20-40KHz to obtain a fecal treatment liquid;
(3) sieving the fecal treatment liquid and water with a 500-mesh sieve according to a volume ratio of 1:1 to obtain fecal residues and filtrate, collecting the filtrate, centrifuging the filtrate at a rotation speed of 2000r/min for 2 minutes, removing supernatant, and collecting precipitate;
(4) adding saturated salt water solution into the precipitate according to the liquid-solid ratio of 2:1, stirring, centrifuging at the rotating speed of 3000r/min for 3 minutes, collecting the floating liquid (supernatant), adding water into the floating liquid according to the volume ratio of 5:1, centrifuging at the rotating speed of 2500r/min for 3 minutes, collecting the centrifugal precipitate, and obtaining the coccidian oocyst.
The coccidian oocysts obtained in example 4 were counted.
Comparative example 1 (glutaraldehyde and pepsin treatment only added)
The method for treating the chicken Eimeria maxima excrement mucus comprises the following steps:
(1) mixing 390kg of chicken Eimeria maxima excrement containing mucus with 130L of water to obtain excrement liquid, adding 8L of glutaraldehyde for mixing, sterilizing, adding phosphoric acid to adjust the pH value to 2.0, adding 5kg of pepsin for mixing, and decomposing the mucus in the excrement liquid to obtain turbid liquid;
(2) sieving the liquid dung and water at a volume ratio of 1:1 with 500 mesh sieve (see figure 11) to obtain liquid dung residue and filtrate, collecting filtrate, centrifuging the filtrate at a rotation speed of 2000r/min for 2 min, discarding supernatant, and collecting precipitate;
(3) adding saturated salt water solution into the precipitate according to the liquid-solid ratio of 2:1, stirring, centrifuging at the rotating speed of 3000r/min for 3 minutes, collecting the floating liquid (supernatant), adding water into the floating liquid according to the volume ratio of 5:1, centrifuging at the rotating speed of 2500r/min for 3 minutes, and collecting the centrifugal precipitate to obtain the coccidian oocysts.
The coccidian oocysts obtained in comparative example 1 were counted.
Comparative example 2 (ultrasonic treatment only)
The method for treating the chicken Eimeria maxima excrement mucus comprises the following steps:
(1) adding feces into water at a solid-to-liquid ratio of 3:1 to obtain feces liquid (which needs to be stored at 2-8 deg.C for 4 hr);
(2) firstly, placing a layer of 1000-mesh nylon filter screen on the inner wall of an ultrasonic processor, then adding 1000g of steel balls into the nylon filter screen, finally adding liquid dung into the nylon filter screen, and carrying out ultrasonic cavitation on finely-divided mucus for 20 minutes under the ultrasonic power of 600W and the frequency of 20-40KHz to obtain a liquid dung treatment liquid;
(3) sieving fecal treatment solution and water at volume ratio of 1:1 with 500 mesh sieve (see figure 12) to obtain fecal residue and filtrate, collecting filtrate, centrifuging the filtrate at 2000r/min for 2 min, removing supernatant, and collecting precipitate;
(4) adding saturated salt water solution into the precipitate according to the liquid-solid ratio of 2:1, stirring, centrifuging at the rotating speed of 3000r/min for 3 minutes, collecting oocyst floating liquid (supernatant), adding water into the supernatant according to the volume ratio of 5:1, centrifuging at the rotating speed of 2500r/min for 3 minutes, and collecting the centrifugal precipitate to obtain the coccidium oocysts.
The coccidian oocysts obtained in comparative example 2 were counted.
COMPARATIVE EXAMPLE 3 (PRIOR ART)
A method for separating coccidian oocysts comprises the following steps:
(1) manually rubbing and kneading the liquid dung, adding water, sieving with a 300-mesh sieve, and collecting residues;
(2) further manually crushing and kneading the residue, adding water, sieving with 500 mesh sieve (see figure 13), repeating for 5 times to obtain feces residue and filtrate, and collecting the filtrate;
(3) centrifuging the filtrate at 2000 rpm for 2 min, discarding the supernatant, and collecting the precipitate;
(4) adding saturated saline solution into the precipitate according to the liquid-solid ratio of 2:1, fully stirring uniformly, centrifuging for 2-3 minutes at 3000 rpm, and collecting oocyst floating liquid;
(5) adding 8 times of volume of water into the supernatant, centrifuging for 2-3 minutes at the rotating speed of 2500 revolutions, and collecting centrifugal precipitates, wherein the precipitates are coccidian oocysts.
The coccidian oocysts of comparative example 3 were counted.
Comparative example 4
The method for treating the chicken Eimeria maxima excrement mucus comprises the following steps:
(1) mixing 390kg of chicken Eimeria maxima excrement containing mucus with 130L of water to obtain excrement liquid, adding 8L of iodine preparation (iodine preparation produced by Zhengdian biotechnology limited of Fushan City) for mixing, sterilizing, adding phosphoric acid to adjust pH to 2.0, adding 5kg of pepsin for mixing, and decomposing mucus in the excrement liquid to obtain turbid liquid (which needs to be stored at 30 ℃ for 4 hours);
(2) firstly, placing a layer of 1000-mesh nylon filter screen on the inner wall of an ultrasonic processor, then adding 1000g of steel balls into the nylon filter screen, finally adding the turbid liquid (stored for 4 hours at the temperature of 30 ℃) into the nylon filter screen, and carrying out ultrasonic cavitation on the fine viscous liquid for 20 minutes under the ultrasonic power of 600W and the frequency of 20-40KHz to obtain the fecal treatment liquid;
(3) sieving fecal treatment solution and water at volume ratio of 1:1 with 500 mesh sieve (see figure 14) to obtain fecal residue and filtrate, collecting filtrate, centrifuging the filtrate at 2000r/min for 2 min, discarding supernatant, and collecting precipitate;
(4) adding saturated salt water solution into the precipitate according to the liquid-solid ratio of 2:1, stirring, centrifuging at the rotating speed of 3000r/min for 3 minutes, collecting oocyst floating liquid (supernatant), adding water into the supernatant according to the volume ratio of 5:1, centrifuging at the rotating speed of 2500r/min for 3 minutes, and collecting the centrifugal precipitate to obtain the coccidium oocysts.
The coccidian oocysts obtained in comparative example 4 were counted.
Comparative example 5
The method for treating the chicken Eimeria maxima fecal mucus comprises the following steps:
(1) mixing 390kg of chicken Eimeria maxima excrement containing mucus with 130L of water to obtain an excrement liquid, then adding phosphoric acid to adjust the pH to 2.0, then adding 5kg of pepsin to mix, and decomposing the mucus in the excrement liquid to obtain a turbid liquid;
(2) firstly, placing a layer of 1000-mesh nylon filter screen on the inner wall of an ultrasonic processor, then adding 1000g of steel balls into the nylon filter screen, finally adding the turbid liquid into the nylon filter screen, and carrying out ultrasonic cavitation on the finely-divided viscous liquid for 20 minutes under the ultrasonic power of 600W and the frequency of 20-40KHz to obtain a fecal treatment liquid;
(3) sieving the fecal treatment liquid and water with a 500-mesh sieve according to a volume ratio of 1:1 to obtain fecal residues and filtrate, collecting the filtrate, centrifuging the filtrate at a rotation speed of 2000r/min for 2 minutes, removing supernatant, and collecting precipitate;
(4) adding saturated salt water solution into the precipitate according to the liquid-solid ratio of 2:1, stirring, centrifuging at the rotating speed of 3000r/min for 3 minutes, collecting oocyst floating liquid (supernatant), adding water into the supernatant according to the volume ratio of 5:1, centrifuging at the rotating speed of 2500r/min for 3 minutes, and collecting the centrifugal precipitate to obtain the coccidium oocysts.
The coccidian oocysts obtained in comparative example 5 were counted.
Comparative example 6
The method for treating the chicken Eimeria maxima fecal mucus comprises the following steps:
(1) mixing 390kg of chicken Eimeria maxima excrement containing mucus with 130L of water to obtain an excrement solution, adding 8L of glutaraldehyde for mixing, sterilizing, and then adding phosphoric acid to adjust the pH value to 2.0 to obtain a turbid solution;
(2) firstly, placing a layer of 1000-mesh nylon filter screen on the inner wall of an ultrasonic processor, then adding 1000g of steel balls into the nylon filter screen, finally adding the turbid liquid into the nylon filter screen, and carrying out ultrasonic cavitation on the finely-divided viscous liquid for 20 minutes under the ultrasonic power of 600W and the frequency of 20-40KHz to obtain a fecal treatment liquid;
(3) sieving the fecal treatment liquid and water with a 500-mesh sieve according to a volume ratio of 1:1 to obtain fecal residues and filtrate, collecting the filtrate, centrifuging the filtrate at a rotation speed of 2000r/min for 2 minutes, removing supernatant, and collecting precipitate;
(4) adding saturated salt water solution into the precipitate according to the liquid-solid ratio of 2:1, stirring, centrifuging at the rotating speed of 3000r/min for 3 minutes, collecting oocyst floating liquid (supernatant), adding water into the supernatant according to the volume ratio of 5:1, centrifuging at the rotating speed of 2500r/min for 3 minutes, and collecting the centrifugal precipitate to obtain the coccidium oocysts.
The coccidian oocysts obtained in comparative example 6 were counted.
The operation method and the counting method for counting the oocysts by the blood counting plate are as follows:
fully and uniformly stirring the coccidian oocyst suspension to be detected, and takingThe sample applicator dropped the sample onto a hemocytometer and counted the total number of sporulated oocysts in the 4 angular squares under a 10X 10 times microscope, multiplied by 2.5X 103This is the sporulated oocyst count in 1ml of oocyst suspension.
In comparative example 1, the residue was filtered in FIG. 11, and the undigested slime was observed. Mixing glutaraldehyde and chicken Eimeria maxima manure liquid containing mucus, sterilizing, adding phosphoric acid to adjust pH to 1.5-2.5, and adding pepsin to decompose mucus; however, the mucus cannot be completely decomposed, and after 500 mesh filtration, the undecomposed mucus still appears, which affects the recovery rate of oocysts.
In comparative example 2 (only ultrasonic treatment is carried out), as shown in the attached figure 12, the feces residue is filtered, a small amount of undecomposed mucus can be seen by naked eyes (the mucus is not decomposed by pepsin), and as glutaraldehyde is not added, even if the feces is stored at the temperature of 2-8 ℃, the feces still can not avoid the mass growth of bacteria in the feces, so that the feces liquid generates a large amount of acid and gas to ferment and expand, and the subsequent working time is increased.
Comparative example 3, see fig. 13 for fecal residue filtration. The existing separation process has the following defects: 1. the fecal matter impurities were reduced when the sieving filtration was repeated 5 times (fig. 7 is a graph of the 5 th filtration concentration of comparative example 3); the feces obtained after the 5 th filtration is observed by microscopy, and more oocysts are still found (see figure 9). However, if the screening and filtering are repeated, the obtained liquid dung is clearer than the 5 th time (see the attached figure 8) and contains little sediment, the liquid dung is centrifuged by a centrifuge, the sediment is suspended in the supernatant, and solid sediment cannot be formed at the bottom of a centrifuge tube, so that oocysts cannot be recovered. Therefore, the number of sieving and filtering is limited, and even if the time cost is not counted, the recovery rate of the oocysts cannot reach a certain level. 2. Since the liquid obtained by filtering is increased correspondingly after the sieving and filtering are repeated for 5 times, the workload of all the subsequent steps is increased correspondingly.
Comparative example 4 with the addition of iodine preparation and pepsin, but with the addition of iodine preparation, which needs to be stored at the temperature of 30 ℃ for 4 hours, the liquid manure can generate a large amount of acid and gas, so that mold growth occurs, and subsequent ultrasonic cavitation of the liquid manure is affected, so that the separation of coccidian oocysts and liquid manure is affected.
In the comparative example 5, the pepsin is added, and the feces are not sterilized by glutaraldehyde, so that the feces still cannot avoid the massive growth of bacteria in the feces even if the feces are stored at the temperature of 2-8 ℃, so that the feces liquid generates a large amount of acid and gas to ferment and expand, and the subsequent working time is increased.
Comparative example 6 glutaraldehyde was added alone, and the mucus was decomposed without pepsin, and a small amount of undecomposed mucus was visible to the naked eye.
Application experiments
The methods of examples 1 to 4 and comparative examples 1 to 6 were applied to the separation of coccidian oocysts, and the time required for the separation and the total number of oocysts obtained were recorded, with the results shown in Table 1.
TABLE 1
Group of | Required time (min) | Amount of oocysts harvested (Yi) | Recovery (%) | Residual state of oocysts of feces and residue after sieving |
Example 1 | 170 | 109.5 | 99.6 | Little residual oocyst (see FIG. 3) |
Example 2 | 174 | 109.0 | 99.1 | Little residual oocyst (see FIG. 4) |
Example 3 | 150 | 105.6 | 96.0 | Little residual oocyst |
Example 4 | 168 | 108.8 | 98.9 | Little residual oocyst |
Comparative example 1 | 186 | 102.5 | 93.2 | The residual oocysts are less (see FIG. 5) |
Comparative example 2 | 210 | 104.0 | 94.6 | Oocysts remained less (see FIG. 6) |
Comparative example 3 | 240 | 77.0 | 70.5 | Oocysts remained much (see FIG. 9) |
Comparative example 4 | 220 | 100.0 | 90.9 | Residual oocysts are less |
Comparative example 5 | 216 | 99.0 | 90.0 | The residual oocysts are less (see FIG. 15) |
Comparative example 6 | 198 | 103.4 | 94.0 | Oocysts remained less (see FIG. 16) |
And (4) supplementary notes: excrement treatment method and McLeod counter counting operation method
1.1 fecal treatment method the collected fecal samples were thoroughly stirred and mixed well.
1.2 McLeod counting operation method adopting random multipoint sampling method (sampling points are not less than 3) to weigh 2g of uniformly stirred feces, placing the feces in a clean beaker, adding 60ml of saturated saline solution, filtering the feces in another clean beaker through a 40-mesh screen after uniform mixing, sucking filtrate after uniform blowing by a rubber head dropper to fill two counting chambers of the McLeod counting plate, standing for 2-5 min, and calculating the total oocyst count (a1) of the two counting chambers under 10 x 10 times of microscope; the sampling operation is repeated once, the total number of oocysts is calculated (a2), and the average value (a) of a1 and a2 is multiplied by 100 to obtain the number of the fecal oocysts (OPG value).
2. The operation method and the counting method for counting the oocysts by using a blood counting plate are as follows: fully and uniformly stirring the coccidian oocyst suspension to be detected, dropping the sample into a blood counting chamber by using a sample injector, counting the total number of sporulated oocysts in 4 angular squares by using a 10X 10-fold microscope, and multiplying the total number by 2.5X 103I.e. in 1ml oocyst suspensionNumber of sporulated oocysts.
3. Oocyst recovery (%) — actual oocyst yield/expected oocyst yield × 100%.
And (4) conclusion:
(1) the time for separating the same amount of feces by the methods of example 1, example 2, example 3, example 4, comparative example 1, comparative example 2, comparative example 4, comparative example 5 and comparative example 6 is respectively shortened by 70 minutes, 66 minutes, 90 minutes, 72 minutes, 54 minutes, 30 minutes, 20 minutes, 24 minutes and 42 minutes compared with the prior art (comparative example 3), and the separation efficiency is improved by 29.2%, 27.5%, 37.5%, 30.0%, 22.5%, 12.5%, 8.3%, 10.0% and 17.5% compared with the prior art; it is shown that the addition of the self-made fecal treatment fluid and the use of ultrasonic treatment in the method of the present invention can significantly shorten the separation time and improve the separation efficiency compared with the prior art, and the combined use effect of the two is more significant in examples 1 to 3 of the present invention.
(2) The recovery rate of the oocysts obtained by separating the same amount of feces by the method used in example 1, example 2, example 3, example 4, comparative example 1, comparative example 2, comparative example 4, comparative example 5 and comparative example 6 is increased by 32.5 hundred million, 32.0 hundred million, 28.6 hundred million, 31.8 hundred million, 25.5 hundred million, 27.0 hundred million, 23.0 hundred million, 22.0 hundred million and 26.4 hundred million compared with the prior art (comparative example 3), and is improved by 41.2%, 40.5%, 36.1%, 40.2%, 32.1%, 34.1%, 28.9%, 27.6% and 33.3% compared with the prior art (comparative example 3); the method has the advantages that the amount of oocysts obtained by adding the self-made fecal treatment fluid and using ultrasonic waves for treatment is remarkably increased compared with the prior art, and the two are combined to have more obvious use effect and higher recovery rate.
(3) From the viewpoint of the fecal treatment process, the treatment method of comparative example 3 is simpler, and only the sieving and filtering need to be simply repeated until the upper limit of the sieving and filtering times is reached. However, because the screening and filtering are repeated here, hidden troubles are buried for the subsequent steps. All filtrate obtained after repeated screening needs to be subjected to centrifugal precipitation, precipitates at the later stage are relatively few, and firm precipitates cannot be well formed at the bottom of a centrifugal tube, so that the final integral precipitates are in a soft state due to reduced viscosity (see figure 10), and all oocysts can be completely floated by repeated floating of saturated salt water. The microscopic examination of the fecal debris oocysts of examples 1 and 2 of the present invention, as shown in FIGS. 3 and 4, showed very little residual oocysts, and the microscopic examination of the fecal debris oocysts of comparative examples 1 and 2, as shown in FIGS. 5 and 6, showed less effect than that of examples 1-2, but relatively little residual oocysts; the residual oocysts were examined under the microscope with the fecal debris of comparative example 3, as shown in FIG. 9, which showed more oocysts. It can be seen that examples 1 to 3 of the present invention have a significant separation efficiency and a significant final recovery rate compared to comparative example 3. Comparative example 4 with the addition of iodine preparation and pepsin, but with the addition of iodine preparation, which needs to be stored at the temperature of 30 ℃ for 4 hours, the liquid manure can generate a large amount of acid and gas, so that mold growth occurs, and subsequent ultrasonic cavitation of the liquid manure is affected, so that the separation of coccidian oocysts and liquid manure is affected.
While the present invention has been described in detail with respect to the methods and applications of the fecal mucus from Eimeria maxima, the principles and embodiments of the present invention are described herein using specific examples, which are set forth only to facilitate an understanding of the methods and their core concepts, including the best mode, and to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The scope of the invention is defined by the claims and may include other embodiments that occur to those skilled in the art. Such other embodiments are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
Claims (10)
1. A method for treating chicken Eimeria maxima excrement mucus is characterized by comprising the following steps:
(1) mixing glutaraldehyde and mucus-containing chicken Eimeria maxima manure liquid, sterilizing, adding phosphoric acid to adjust pH to 1.5-2.5, and adding pepsin to decompose mucus in the manure liquid to obtain turbid liquid;
(2) and processing the turbid liquid by ultrasonic cavitation to obtain the fecal treatment liquid.
2. The process according to claim 1, wherein the step (2) further comprises sieving the fecal treatment fluid, collecting the filtrate, centrifuging the filtrate again, and collecting the precipitate obtained by the centrifugation; adding saturated saline solution into the precipitate, stirring, centrifuging, floating, collecting supernatant, adding water, centrifuging, and collecting the precipitate to obtain chicken Eimeria maxima oocysts.
3. The process according to claim 1, characterized in that in step (1), the liquid-solid ratio of glutaraldehyde and pepsin is (8-16): (5-10) ml/g.
4. The method as claimed in claim 1, wherein in step (1), the fecal sewage is prepared from chicken Eimeria maxima feces containing mucilage and water according to a solid-to-liquid ratio of (1-3): (0.9-0.93) g/mL.
5. The treatment method according to claim 1, wherein in the step (2), the specific operation process of the ultrasonic cavitation treatment is as follows: firstly, a layer of 800-plus-1200-mesh nylon filter screen is placed on the inner wall of the ultrasonic treatment machine, then, 1000-plus-1100 g of steel balls are added into the nylon filter screen, finally, the turbid liquid is added into the nylon filter screen, and ultrasonic treatment is carried out for 10-30 minutes under the ultrasonic power of 300-plus-600W and the frequency of 20-40 KHz.
6. The process according to claim 2, wherein the number of said sifting is from 1 to 5; the screening process is that the fecal treatment fluid and water are mixed according to the volume ratio of 1 (1-2), and then screened by a 200-mesh and 500-mesh screen.
7. The process as claimed in claim 2, wherein the rotation speed of the centrifugation is 2000-3000r/min, and the time of the centrifugation is 1-5 min.
8. The treatment method according to claim 2, wherein the liquid-solid ratio of the saturated brine to the precipitate is (1-3): 1L/kg.
9. The treatment method according to claim 2, wherein the volume ratio of the supernatant to the water is 1 (5-10).
10. Use of a treatment according to any one of claims 1 to 9 for the isolation of oocysts from parasites on animals.
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