Epidemic prevention support device, epidemic prevention support method and program

Abstract

To provide an epidemic prevention assisting device, an epidemic prevention assisting method and a program which can assist in taking effective preventive measures against epidemic animal diseases.SOLUTION: An epidemic prevention assisting device has a presentation part that presents preventive measures against epidemics on the basis of data indicating the infectious dose of a pathogen causing an animal disease in individual domestic animals and information on a farm in which the individual domestic animals are bred.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to an epidemic prevention support device, an epidemic prevention support method, and a program. [Background technology]

[0002] Livestock infectious diseases such as African swine fever, foot-and-mouth disease, and avian influenza not only cause enormous economic losses to the livestock industry, but also pose a serious problem in terms of ensuring a stable food supply for humanity. Early detection and early countermeasures for infectious diseases are essential to control the outbreak of infectious diseases in livestock.

[0003] Infectious bovine lymphoma (EBL) is one of the major livestock infectious diseases currently spreading in Japan. While demand for EBL testing is increasing, the public testing institutions, the Livestock Hygiene Centers, are facing major challenges in terms of budget and human resource shortages. Testing is also available through private testing companies, but the cost is high and it usually takes about a week to receive the test results, so few producers use private testing companies. Furthermore, even if they are able to undergo testing, many farms do not use the test results in their disease prevention measures. Establishing comprehensive technology or systems that can support the implementation of effective disease prevention measures against EBL is an urgent issue.

[0004] Patent document 1 discloses a device that provides indicators for local governments and farms involved in animal infectious disease control to make decisions during the work stage of prevention measures, and visualizes risk information related to animal infectious diseases. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-199556 Summary of the Invention [Problem to be solved by the invention]

[0006] The device disclosed in Patent Document 1 uses a Markov chain model to calculate the probability of spatial spread of an animal infectious disease to other farms in a certain area, for example, and identifies farms where epidemiological testing should be conducted based on the calculated infection probability. While the device provides risk information based on a simulation, it cannot provide disease prevention measures based on the actual infection status of livestock infectious diseases on a farm. The disease prevention measures that can be implemented on a farm may be limited by the farm's rearing environment.

[0007] The present invention has been made in view of the above circumstances, and aims to provide a quarantine support device, a quarantine support method, and a program that can support the implementation of effective quarantine measures against livestock infectious diseases. [Means for solving the problem]

[0008] The epidemic prevention support device according to a first aspect of the present invention comprises: cattle In individuals bovine infectious lymphoma virus Data showing the infectious dose of cattle A presentation unit is provided that presents quarantine measures based on information about the farm where the individual is raised. 、 The information about the farm is The information includes at least one of whether or not cattle individuals can be culled or replaced, whether or not they can be kept in isolation, whether or not they can be kept in tandem, and whether or not newborn cattle individuals can be kept in isolation, The data showing the infectious dose is Includes proviral copy number quantified by droplet digital PCR. The primers used in the droplet digital PCR are It contains a forward primer whose base sequence is shown in SEQ ID NO: 1 and a reverse primer whose base sequence is shown in SEQ ID NO: 2. .

[0012] before The primers used for the droplet digital PCR are: A forward primer having a base sequence shown in SEQ ID NO: 9 and a reverse primer having a base sequence shown in SEQ ID NO: 10 were used. moreover include, This may also be the case.

[0013] Moreover, the presentation unit The aforementioned cattlein the individual's blood The bovine infectious lymphoma virus The prevention measures are proposed based on the presence or absence of specific antibodies against the virus. This may also be the case.

[0014] Moreover, the presentation unit The aforementioned cattle The individual bovine infectious lymphoma virus Present disease prevention measures based on susceptibility to This may also be the case.

[0015] Moreover, the presentation unit The aforementioned cattle The individual has bovine infectious lymphoma virus The gene sequence of cattle Other than individuals cattle The above-mentioned detected from an individual bovine infectious lymphoma virus Compared with the gene sequence of cattle The individual bovine infectious lymphoma virus Provides information to estimate the route of infection, This may also be the case.

[0016] A quarantine support method according to a second aspect of the present invention comprises: cattle In individuals bovine infectious lymphoma virus Data showing the infectious dose of cattle The method includes a step of suggesting disease prevention measures based on information about the farm where the individual is raised. fruit, The information about the farm is The information includes at least one of whether or not cattle individuals can be culled or replaced, whether or not they can be kept in isolation, whether or not they can be kept in tandem, and whether or not newborn cattle individuals can be kept in isolation, The data showing the infectious dose is Includes proviral copy number quantified by droplet digital PCR. The primers used in the droplet digital PCR are The primer comprises a forward primer having a base sequence shown in SEQ ID NO: 1 and a reverse primer having a base sequence shown in SEQ ID NO: 2. nothing.

[0017] A program according to a third aspect of the present invention comprises: Computer, cattle In individuals bovine infectious lymphoma virus Data showing the infectious dose of cattle It functions as a presentation unit that suggests disease prevention measures based on information about the farm where the individual is raised.、 The information about the farm is The information includes at least one of whether or not cattle individuals can be culled or replaced, whether or not they can be kept in isolation, whether or not they can be kept in tandem, and whether or not newborn cattle individuals can be kept in isolation, The data showing the infectious dose is Includes proviral copy number quantified by droplet digital PCR. The primers used in the droplet digital PCR are It contains a forward primer whose base sequence is shown in SEQ ID NO: 1 and a reverse primer whose base sequence is shown in SEQ ID NO: 2. . [Effects of the Invention]

[0018] According to the present invention, it is possible to support the implementation of effective prevention measures against infectious diseases in livestock. [Brief explanation of the drawings]

[0019] [Figure 1] 1A and 1B are diagrams illustrating the configuration of an epidemic prevention support device according to an embodiment of the present invention, in which (A) is a block diagram illustrating the hardware configuration of the epidemic prevention support device, and (B) is a block diagram illustrating the functions of the epidemic prevention support device. [Figure 2] FIG. 10 is a diagram illustrating farm data. [Figure 3] FIG. 10 is a diagram illustrating an example of data to be analyzed. [Figure 4] 2 is a diagram showing epidemic prevention measures (tasks) presented by the epidemic prevention support device of FIG. 1. FIG. [Figure 5] 1. FIG. 4 is a flowchart showing an epidemic prevention measure presentation process performed by the epidemic prevention support device of FIG. [Figure 6] 1. FIG. 4 is a flowchart showing a process for selecting quarantine measures for high-risk cattle performed by the quarantine support device of FIG. [Figure 7] FIG. 2 is a flowchart showing an information presentation process performed by the epidemic prevention support device of FIG. [Figure 8] FIG. 1 is a diagram showing the results of examining the annealing temperature in Test Example 1. [Figure 9] FIG. 1 shows the results of simultaneous measurement of the env gene of BLV provirus and the bovine RPP30 gene in Test Example 1. [Figure 10]FIG. 1 is a graph showing the correlation between the amount of provirus quantified by real-time PCR (Polymerase Chain Reaction) and the amount of provirus quantified by droplet digital (dd) PCR in Test Example 1. [Figure 11] FIG. 1 shows bands obtained by electrophoresis in Test Example 2. [Figure 12] This figure shows the results of tests A, B, C, and D related to Test Example 3. A1, A2, and A3 show the amplification curves of samples 1, 2, and 3, respectively, in test A. B1, B2, and B3 show the signals of samples 1, 2, and 3, respectively, in test B. C1, C2, and C3 show the signals of samples 1, 2, and 3, respectively, in test C. D1, D2, and D3 show the signals of samples 1, 2, and 3, respectively, in test D. DETAILED DESCRIPTION OF THE INVENTION

[0020] Embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0021] (Embodiment) A disease prevention support device 100 according to this embodiment will be described. The disease prevention support device 100 is an information processing device useful for planning feasible disease prevention measures against livestock infectious diseases. Livestock include agricultural animals, pet animals, and laboratory animals, and are particularly agricultural animals. Specifically, livestock include cows, buffalo, sheep, goats, pigs, horses, dogs, cats, rabbits, camels, llamas, alpacas, reindeer, donkeys, mink, ferrets, hamsters, mice, rats, guinea pigs, chickens, pigeons, turkeys, quails, guinea fowl, ducks, geese, carp, goldfish, silkworms, and honeybees. Preferably, the livestock are cows, pigs, or chickens.

[0022] Livestock infectious diseases are diseases caused by infection with pathogens such as viruses, mycoplasmas, rickettsiae, fungi, protozoa, and parasites, and are transmissible from one livestock individual to another. Livestock infectious diseases include rinderpest, bovine pleuropneumonia, foot-and-mouth disease, epidemic encephalitis, rabies, vesicular stomatitis, Rift Valley fever, anthrax, hemorrhagic septicemia, brucellosis, tuberculosis, Johne's disease, piroplasmosis, anaplasmosis, transmissible spongiform encephalopathy, glanders, equine infectious anemia, African horse sickness, swine fever, African swine fever, swine vesicular disease, fowl cholera, highly pathogenic avian influenza, Newcastle disease, poultry salmonellosis, maggot disease, brut ing, Akabane disease, malignant catarrhal fever, Chuzan disease, lumpy skin disease, bovine viral diarrhea, infectious bovine rhinotracheitis, EBL, Aino virus infection, Ibaraki disease, bovine papular stomatitis, bovine ephemeral fever, melioidosis, tetanus, black jaundice, leptospirosis, salmonellosis, bovine campylobacteriosis, trypanosomiasis, trichomoniasis, neosporosis, bovine gnat larvae disease, Nipah virus infection, equine influenza, equine Viral arteritis, equine rhinopneumonitis, Hendra virus infection, horsepox, tularemia, contagious equine metritis, equine paratyphoid, pseudomyelitis, peste des petit ruminants, contagious pustular dermatitis, Nairobi sheep disease, sheep pox, Maedi-visna, contagious agalactia, epidemic ovine abortion, toxoplasmosis, mange, goat pox, caprine arthritis and encephalitis, contagious caprine pleuropneumonia, Aujeszky's disease, transmissible gastroenteritis, porcine tesseoviral encephalomyelitis, porcine reproductive and respiratory disorders These include swine vesicular exanthema, porcine epidemic diarrhea, atrophic rhinitis, swine erysipelas, swine dysentery, avian influenza, fowl pox, Marek's disease, avian infectious bronchitis, avian infectious laryngotracheitis, infectious bursal disease of Fabricius, avian leukosis, avian tuberculosis, avian mycoplasmosis, leucocytozoonosis, duck viral hepatitis, duck viral enteritis, rabbit hemorrhagic disease, rabbit myxoma, varroasis, chalk disease, acarinosis, and nosematosis.

[0023] In the following, this embodiment will be described assuming that the livestock infectious disease is EBL. EBL is caused by the bovine infectious lymphoma virus (BLV). The number of EBL cases in Japan is on the rise. BLV primarily infects B cells and is incorporated into the host's DNA as a provirus, resulting in persistent infection. As the disease progresses, 2-3% of BLV-infected cattle develop B-cell lymphoma, or EBL. There is no vaccine for BLV or treatment for EBL, and cattle that develop EBL are discarded. BLV-infected cattle experience reduced milk production and reduced immunity. Currently, the only way to prevent the spread of infection is to detect BLV-infected cattle and cull or isolate them.

[0024] As shown in FIG. 1(A), the epidemic prevention support device 100 has a configuration in which a memory unit 10, a RAM (Random Access Memory) 20, an input device 30, a display device 40, and a CPU (Central Processing Unit) 50 are connected by a bus 60.

[0025] The storage unit 10 includes a non-volatile storage medium such as a read-only memory (ROM), a hard disk drive (HDD), a flash memory, etc. The storage unit 10 stores farm data 11 and a presentation program 12 in addition to various data and software programs.

[0026] Farm data 11, which is information about farms where cattle are raised, includes data indicating, for example, whether cattle can be culled or replaced, whether isolation rearing is possible, whether tether rearing is possible, and whether newborn calves can be kept in isolation. The farm data 11 illustrated in FIG. 2 is a table in which, for each of multiple farms F1, F2, and F3, "Yes" is assigned for cattle culling or replacement, isolation rearing, tether rearing, and isolation rearing of newborn calves if possible, and "No" if not possible. Isolation rearing refers to rearing one or more cattle in an independent barn (isolation barn) isolated from the rest of the herd. When isolation rearing is possible, for example, by moving infected cattle from a herd that had been reared in a free-stall barn to an isolation barn, the transmission of BLV from infected cattle to non-infected cattle can be prevented. Because newborn calves are smaller than adult cattle, an isolation barn with a smaller capacity than that required for adult cattle can be used for newborn calves. Tethered housing is the practice of keeping cattle in a tethered barn. When tethered housing is possible, for example, by moving infected cattle to a tethered barn, contact between infected cattle and non-infected cattle kept in free-stall barns can be avoided.

[0027] 1A, the RAM 20 functions as a main memory for the CPU 50, and when the CPU 50 executes the presentation program 12, the presentation program 12 is loaded into the RAM 20. Data input via the input device 30 is temporarily stored in the RAM 20.

[0028] The input device 30 is hardware that allows a user to input data into the epidemic prevention support device 100. The data input via the input device 30 includes data indicating the infection dose of BLV, which causes EBL in cattle. The infection dose of BLV is, for example, the provirus copy number. The provirus copy number can be quantified by known techniques, such as real-time PCR or ddPCR. PCR can be performed using primers that target the BLV env gene, pol gene, or the like. Alternatively, the provirus copy number can be quantified by multiplex PCR, which uses multiple primer pairs to amplify multiple gene regions.

[0029] The data input via the input device 30 includes farm data 11, the number of detected provirus copies for each cow, data indicating the presence or absence of specific antibodies against BLV in the blood of the multiple cows, data regarding the susceptibility of the multiple cows to BLV, and genetic sequence data of BLV possessed by each cow, etc. The data input via the input device 30 is stored in the memory unit 10.

[0030] Data indicating the presence or absence of BLV-specific antibodies in blood is, for example, data indicating whether specific antibodies were detected (positive) or not (negative) by enzyme-linked immunosorbent assay (ELISA). BLV-specific antibodies can be detected using a commercially available ELISA kit (e.g., manufactured by JNC). An example of a specific antibody is anti-BLV Env gp51 antibody contained in bovine serum.

[0031] Data on susceptibility to BLV is, for example, data indicating the presence or absence of a pathogenic resistance gene or a pathogenic susceptibility gene. The presence or absence of a pathogenic resistance gene or a pathogenic susceptibility gene can be determined by PCR-RFLP (Restriction Fragment Length Polymorphism). By PCR-RFLP, for example, the presence or absence of susceptibility or resistance to BLV can be determined depending on the MHC class II DRB3 allele type. The MHC class II DRB3 allele type can be determined using restriction enzymes Rsa I, Hae III, and BstY I.

[0032] The BLV gene sequence data is obtained by a known sequence analysis method. The gene sequence data for multiple BLVs detected from multiple cattle may be stored in advance in the storage unit 10. In this case, the gene sequence data is stored in the storage unit 10 as a database that can be compared with gene sequence data input as the analysis target.

[0033] 3 shows an example of data to be analyzed that is input via the input device 30. The data is such that the IDs (1 to 32) assigned to each individual cow raised on the farm correspond to the number of copies of the provirus, data indicating the presence or absence of specific antibodies, and data indicating the presence or absence of susceptibility to BLV.

[0034] The display device 40 is a display for outputting the results of data analysis by the CPU 50. The CPU 50 reads the presentation program 12 stored in the storage unit 10 into the RAM 20 and executes the presentation program 12 to realize the functions described below.

[0035] 1, (B) of FIG. 1 is a block diagram showing the functions realized by the CPU 50. The presentation program 12 causes the CPU 50 to realize the functions of the presentation unit 1 and the output unit 2.

[0036] The display unit 1 displays disease prevention measures based on the BLV provirus copy number and farm data 11 related to the farm where the cattle are raised. Specifically, the display unit 1 displays disease prevention measures that can be implemented on the farm, based on the provirus copy number, with reference to the farm data 11. Furthermore, the display unit 1 displays disease prevention measures based on the presence or absence of specific antibodies and susceptibility to BLV, in addition to the provirus copy number.

[0037] Figure 4 shows the disease prevention measures (actions) suggested by the display unit 1. The higher the provirus copy number (e.g., 500 copies or more per 50 ng of DNA), the higher the risk of BLV transmission from one cow to another. For example, display unit 1 suggests disease prevention measures #1 to #4 for cows with a provirus copy number of less than 500, depending on the presence or absence of specific antibodies. On the other hand, for cows with a provirus copy number of 500 or more (high-risk cows), display unit 1 suggests disease prevention measures #5 to #12 based on farm data 11, the presence or absence of specific antibodies, and, in some cases, susceptibility to BLV. Note that the "constitution" in measures #11 and #12 refers to the ease of BLV proliferation in the body, for example, the presence or absence of susceptibility to BLV determined by the DRB3 allele type described above.

[0038] The presentation unit 1 associates the presented disease prevention measures with the IDs of the cows and stores them in the memory unit 10. The presentation unit 1 inputs the disease prevention measures and the IDs of the cows associated with the disease prevention measures to the output unit 2. The output unit 2 displays the input disease prevention measures and the IDs associated with the disease prevention measures via the display device 40. As a result, the display device 40 displays the disease prevention measures and the IDs of the cows associated with the disease prevention measures.

[0039] The display unit 1 compares the genetic sequence of the BLV possessed by the cow with the genetic sequence of BLV detected in other cows and displays information for estimating the route of infection of the cow with BLV. In comparing the genetic sequences, the display unit 1 determines, for example, whether the BLV types match or whether the BLVs are closely related, using phylogenetic tree analysis. Phylogenetic tree analysis can be performed by known methods. The display unit 1 executes a phylogenetic tree analysis software program stored in the memory unit 10 to obtain a phylogenetic tree constructed based on the evolutionary distance between BLVs determined from the BLV genetic sequences. Each BLV in the phylogenetic tree is associated with the ID of the cow in which that BLV was detected. The output unit 2 displays the phylogenetic tree on the display device 40 so that the ID of the cow in which the BLV being analyzed was detected can be identified.

[0040] The quarantine countermeasure presentation process by the quarantine support device 100 will be explained in more detail with reference to the flowcharts shown in Figures 5 and 6. Here, it is assumed that cows with IDs 1 to 32 shown in Figure 3 are raised on farm F, which is not illustrated in Figure 2. It is assumed that the user has previously obtained the data shown in Figure 3 for the cows with IDs 1 to 32. The memory unit 10 stores farm data 11 related to farm F that has been input in advance by the user. The memory unit 10 also stores "N," which will be described later, and N = 0 before the start of the quarantine countermeasure presentation process.

[0041] When a user inputs the data shown in FIG. 3 into the epidemic prevention support device 100 via the input device 30 and issues an instruction to start the epidemic prevention countermeasure presentation process, the epidemic prevention support device 100 starts the epidemic prevention countermeasure presentation process. As shown in FIG. 5, the presentation unit 1 stores the input data in the memory unit 10 and counts the number of IDs Nt (step S1). Next, the presentation unit 1 adds 1 to N (step S2). The presentation unit 1 references the data stored in the memory unit 10 and acquires data on cows whose IDs match N (step S3). The presentation unit 1 determines whether the specific antibody in the acquired cow data is positive or not (step S4). If the specific antibody is not positive, i.e., negative (step S4; No), the presentation unit 1 determines whether the copy number of the provirus is one or more (step S5). If the copy number of the provirus is not 1 or more, i.e., if the copy number is 0 (step S5; No), the display unit 1 stores N (= ID) in association with #1 in the memory unit 10 (step S6). If the copy number of the provirus is 1 or more (step S5; Yes), the display unit 1 stores N in association with #2 in the memory unit 10 (step S7).

[0042] If the specific antibody is positive (Step S4; Yes), the display unit 1 determines whether the provirus copy number is 1 or more (Step S8). If the provirus copy number is 0 (Step S8; No), the display unit 1 stores N in the memory unit 10 in association with #3 (Step S9). If the provirus copy number is 1 or more (Step S8; Yes), the display unit 1 determines whether the provirus copy number is less than 500 (Step S10). If the provirus copy number is less than 500 (Step S10; Yes), the display unit 1 stores N in the memory unit 10 in association with #4 (Step S11).

[0043] If the provirus copy number is 500 or more (Step S10; No), the presentation unit 1 performs a process for selecting quarantine measures for high-risk cattle (Step S12). Here, the process for selecting quarantine measures for high-risk cattle will be described with reference to FIG. 6. The presentation unit 1 refers to the farm data 11 to determine whether or not cattle can be culled or updated (Step S21). If cattle can be culled or updated (Step S21; Yes), the presentation unit 1 stores N in the memory unit 10 in association with #5 (Step S22). Next, the presentation unit 1 determines whether or not there is data indicating susceptibility in the cattle data (Step S23). If there is data indicating susceptibility (Step S23; Yes), the presentation unit 1 determines whether or not the cattle are susceptible (Step S24). If the cattle are susceptible (Step S24; Yes), the presentation unit 1 stores N in the memory unit 10 in association with #9 (Step S25), and the process for selecting quarantine measures for high-risk cattle ends. On the other hand, if there is no susceptibility, i.e., there is resistance (step S24; No), the presentation unit 1 stores N in the memory unit 10 in association with #10 (step S26), and ends the process of selecting quarantine measures for high-risk cattle. If there is no data indicating susceptibility (step S23; No), the presentation unit 1 stores N in the memory unit 10 in association with #11 (step S27), and ends the process of selecting quarantine measures for high-risk cattle.

[0044] If culling or replacement of cattle is not possible (Step S21; No), the presentation unit 1 refers to the farm data 11 and determines whether isolation rearing is possible (Step S28). If isolation rearing is possible (Step S28; Yes), the presentation unit 1 stores N in the memory unit 10 in association with #6 (Step S29), and ends the high-risk cattle quarantine measure selection process. If isolation rearing is not possible (Step S28; No), the presentation unit 1 refers to the farm data 11 and determines whether or not confined rearing is possible (Step S30). If confined rearing is possible (Step S30; Yes), the presentation unit 1 stores N in the memory unit 10 in association with #7 (Step S31), and ends the high-risk cattle quarantine measure selection process. If confined rearing is not possible (Step S30; No), the presentation unit 1 refers to the farm data 11 and determines whether or not isolation rearing of newborn calves is possible (Step S32). If it is possible to keep the newborn calf in isolation (step S32; Yes), the presentation unit 1 stores N in the storage unit 10 in association with #8 (step S33), and the process of selecting quarantine measures for high-risk cattle is completed.

[0045] If isolation of the newborn calf is not possible (Step S32; No), the presentation unit 1 determines whether or not there is data indicating susceptibility in the cow's data (Step S34). If there is data indicating susceptibility (Step S34; Yes), the presentation unit 1 stores N in the memory unit 10 in association with #12 (Step S35), and ends the process of selecting quarantine measures for high-risk cattle. If there is no data indicating susceptibility (Step S34; No), the presentation unit 1 stores N in the memory unit 10 in association with #11 (Step S27), and ends the process of selecting quarantine measures for high-risk cattle.

[0046] Returning to FIG. 5, following steps S6, S7, S9, S11, and S12, the presentation unit 1 determines whether N matches Nt (step S13). If N differs from Nt (step S13; No), the presentation unit 1 returns to step S2. If N matches Nt (step S13; Yes), the output unit 2 refers to the storage unit 10 and displays N and the quarantine measures (#1 to #12) associated with N on the display device 40 (step S14). As a result, N, i.e., the cow's ID and the quarantine measures, are displayed on the display device 40. The quarantine support device 100 ends the quarantine measure presentation process.

[0047] Estimating the infection route is useful for disease prevention measures. In accordance with a user's instruction, the presentation unit 1 executes an information presentation process that presents information for estimating the infection route. The information presentation process will be described with reference to FIG. 7. The memory unit 10 pre-stores genetic sequence data of BLV carried by multiple infected cattle raised on farm F.

[0048] The presentation unit 1 waits until an instruction is received from the user (step S41; No). When the user inputs data instructing information presentation processing via the input device 30 (step S41; Yes), the presentation unit 1 executes a phylogenetic tree analysis software program and acquires a phylogenetic tree (step S42). The output unit 2 displays the phylogenetic tree on the display device 40 (step S43). The epidemic prevention support device 100 ends the information presentation processing.

[0049] The epidemic prevention support device 100 according to this embodiment presents epidemic prevention measures based on the copy number of the BLV provirus and farm data 11 relating to the farm F where cattle are raised. This makes it possible to support the implementation of effective epidemic prevention measures against BLV. This allows users and producers (livestock owners) to plan optimal epidemic prevention measures that prioritize feasibility.

[0050] The farm data 11 may include at least one of whether cattle can be culled or replaced, whether isolation rearing is possible, whether tether rearing is possible, and whether newborn calves can be kept in isolation. In the process of selecting quarantine measures for high-risk cattle, the presentation unit 1 presents the measures in order of increasing preventive effectiveness, such as culling or replacement of cattle → isolation rearing → tether rearing → isolation rearing of newborn calves, so that quarantine measures that are feasible for each farm and have the highest preventive effectiveness can be presented. Note that the farm data 11 is not limited to data indicating whether cattle can be culled or replaced, whether isolation rearing is possible, whether tether rearing is possible, and whether newborn calves can be kept in isolation, but may also include, for example, farm size, animal husbandry hygiene management methods, details of quarantine measures being implemented, geographical conditions of the farm, type of feed, people and vehicles entering and leaving the farm, handling of compost, and whether wild animals are invading the farm.

[0051] Note that the quarantine measures shown in Figure 4 are merely examples and are not limited to these. The display unit 1 may display quarantine measures according to the provirus load. For example, the display unit 1 may display a quarantine measure of preferentially culling or replacing infected cattle whose provirus copy number exceeds a threshold, and a quarantine measure of preferentially isolating and rearing infected cattle whose provirus copy number exceeds a threshold. Furthermore, in order to prevent vertical infection, the display unit 1 may display a quarantine measure of not allowing infected cattle whose provirus copy number exceeds a threshold to produce calves.

[0052] The data indicating the infection amount includes the number of provirus copies quantified by ddPCR, etc. Because ddPCR can absolutely quantify the amount of BLV with high accuracy, the epidemic prevention support device 100 can propose feasible and highly effective epidemic prevention measures based on highly accurate information on infection risk.

[0053] The display unit 1 suggests disease prevention measures based on the presence or absence of specific antibodies against BLV in the blood. This allows the presence or absence of BLV infection to be utilized in disease prevention measures. Furthermore, by combining the presence or absence of specific antibodies with the provirus copy number, it is possible to suggest to the user the influence of maternal antibodies or the possibility that the infection is below the detection limit. Based on the presence or absence of specific antibodies, the display unit 1 may display the infection history on the display device 40 via the output unit 2, or may suggest vaccination as a disease prevention measure.

[0054] The indicator 1 suggests disease prevention measures based on BLV susceptibility. This allows the indicator 1 to suggest cattle that should be prioritized for culling, replacement, and retention, enabling users to implement efficient disease prevention measures.

[0055] The display unit 1 compares the genetic sequence of BLV carried by the cow with genetic sequences of BLV detected in other cows and displays information for estimating the route by which the cow became infected with BLV. By estimating the route of infection, including the source of infection, users can take disease prevention measures such as halting the introduction of cattle from the farm that is the source of infection and conducting thorough testing before introduction. The display unit 1 may also display epidemiological associations between farms by combining data on the movement history of cattle between farms with the results of phylogenetic tree analysis.

[0056] Furthermore, the display unit 1 may determine whether the specific antibody is positive or not after determining whether the provirus copy number is 1 or more. If the provirus copy number is 0 and the specific antibody is negative, the display unit 1 stores N in association with #1 in the memory unit 10, and if the provirus copy number is 1 or more and the specific antibody is negative, the display unit 1 stores N in association with #2 in the memory unit 10. If the provirus copy number is 0 and the specific antibody is positive, the display unit 1 stores N in association with #3 in the memory unit 10, and if the provirus copy number is 1 or more and the specific antibody is positive, the display unit 1 executes step S10 and subsequent steps.

[0057] The epidemic prevention support device 100 may function as a server on a network such as the Internet. In this case, for example, a producer can access the epidemic prevention support device 100 via the Internet and input an ID that can identify an individual cow, thereby viewing the epidemic prevention measures associated with the ID stored in the storage unit 10. There may be multiple farms, and in response to access using identification information unique to the farm, the epidemic prevention support device 100 may transmit to the producer's terminal via the Internet information such as the test results of the cattle raised on the farm and the presented epidemic prevention measures, as well as the trend in the infection rate on the farm (the number of high-risk cattle relative to the number of cattle raised on the farm), a ranking of the transmission risk based on the copy number of the provirus in infected cattle, the test history, and the IDs of untested cattle.

[0058] The presentation program 12 and various software programs used in the epidemic prevention support device 100 can be stored and distributed on computer-readable recording media such as CD-ROMs (Compact Disc Read Only Memory), DVDs (Digital Versatile Discs), Magneto-Optical Discs, USB (Universal Serial Bus) memory, memory cards, and HDDs. By installing the presentation program 12 and various software programs on a specific or general-purpose computer, the computer can function as the epidemic prevention support device 100. Alternatively, the presentation program 12 and various software programs may be stored in a storage device owned by another server on the Internet, and the presentation program 12 and various software programs may be downloaded from the server.

[0059] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples. [Example]

[0060] (Test Example 1: Quantification of BLV provirus by ddPCR) Blood samples were collected from 139 cows diagnosed with BLV infection at a Wagyu cattle farm in Miyazaki Prefecture. DNA was extracted using MagDEA Dx SV (Precision System Science) and magLEAD 12gC (Precision System Science). The concentration of the extracted DNA was measured using a NanoDrop 8000 (Thermo Fisher Scientific).

[0061] The BLV proviral load (proviral copy number / 50 ng DNA) was measured by real-time PCR using a bovine infectious lymphoma virus detection probe / primer / positive control (Takara Bio) and Cycleave PCR™ Reaction Mix SP (Takara Bio) on an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems).

[0062] To detect the BLV provirus by ddPCR, primers and probes were designed targeting the BLV env gene. The nucleotide sequences of the forward primer, reverse primer, and probe targeting the env gene are shown in SEQ ID NOs: 1, 2, and 3, respectively. The probe for the env gene was labeled with FAM. To calculate the BLV infection rate, the bovine RPP30 gene was measured as a housekeeping gene. The nucleotide sequences of the forward primer, reverse primer, and probe targeting the RPP30 gene are shown in SEQ ID NOs: 4, 5, and 6, respectively. The probe for the RPP30 gene was labeled with HEX.

[0063] The PCR reaction mixture (20 μl) consisted of 10 μl of 2× ddPCR Supermix, 1 μl of primer (env gene), 1 μl of primer (RPP30 gene), 2 μl of template, and 6 μl of water. Thermal cycling consisted of an initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 94°C for 30 seconds and 51°C for 1 minute, followed by 98°C for 10 minutes and a hold time of 8°C.

[0064] Droplets were prepared using a QX-200™ droplet generator (Bio-Rad). PCR reactions were performed using a CFX96™ thermal cycler (Bio-Rad). After PCR, the number of droplets was measured using a QX200™ droplet reader (Bio-Rad). The proviral load of BLV (proportion of infected cells) was calculated using the following formula: Proviral load = (BLV copy number / (RPP30 copy number / 2)) × 100

[0065] To analyze the correlation between real-time PCR and ddPCR measurements, Spearman's rank correlation coefficient was used, and a test was performed at a significance level of 5%.

[0066] (result) When examining annealing temperature conditions using a BLV temperature gradient (50-63°C), the best results were obtained at 50°C or 51°C, as shown in Figure 8. Simultaneous measurement of two genes, the BLV provirus (env gene) and the RPP30 gene, in one well yielded strong signals for both the env gene and the RPP30 gene, clearly distinguishing between positive and negative results, as shown in Figure 9. The amount of BLV provirus was calculated and analyzed for correlation with real-time PCR, confirming a significant correlation between the two, as shown in Figure 10.

[0067] (Test Example 2: Discrimination of DRB3 allele type by PCR-RFLP method) DNA is extracted using the Wizard™ Genomic DNA Purification Kit (Promega) as follows. 1. Add 900 μl of Cell Lysis Solution and 300 μl of blood sample to a 1.5 mL tube and mix by inverting. 2. Let stand for 10 minutes. 3. Centrifuge at 14,000 x g for 20 seconds. 4. Discard the supernatant and then vortex the pellet. 5. Add 300 μl of Nuclei Lysis Solution, pipette gently, and then mix by inverting. 6. Leave at 37°C for 30 minutes. 7. Add 100 μl of Protein Precipitation Solution and vortex for 20 seconds. 8. Centrifuge at 14,000 x g for 3 minutes. 9. Aspirate the supernatant and transfer it to a 1.5 mL flat-bottom tube containing 300 μl of 100% isopropanol, then mix by inversion. 10. Centrifuge at 14,000 x g for 1 minute. 11. Decant the supernatant, add 300 μl of 70% ethanol, and centrifuge at 14,000 × g for 1 minute. 12. Aspirate the supernatant using a 200 μl pipette and leave the lid open to air dry. 13. Add 100 μl of sterile water and leave to stand at 65°C for 1 hour or at 4°C overnight. 14. Measure DNA concentration using NanoDrop 8000.

[0068] Next, the BoLA-DRB3 exon 2 region was amplified by PCR. The following reaction mixture was prepared using the TaKaRa Ex Taq Hs kit (Takara Bio Inc.). The base sequences of primers (HL030) and (HL032) are shown in SEQ ID NOs: 7 and 8, respectively. The reaction mixture consisted of 14.5 μl of water, 2 μl of 10× buffer, 2 μl of dNTPs, 0.2 μl of 10 μM primer (HL030), 0.2 μl of 10 μM primer (HL032), 0.1 μl of Ex Taq Hs, and 1 μl of template (DNA sample).

[0069] The reaction mixture was placed in a 0.2 ml PCR tube and reacted in a thermal cycler with the following thermal cycle: 94°C for 10 minutes, followed by 35 cycles of 94°C for 30 seconds, 60°C for 15 seconds, and 72°C for 30 seconds, followed by 72°C for 10 minutes and a 4°C hold.

[0070] The PCR product was digested with the restriction enzyme BstYI (BioLabs) and the DRB3 allele type was determined based on the size of the digested fragments detected by electrophoresis: The reaction mixture consisted of 10 μl of PCR product, 0.5 μl of BstYI (10,000 U / ml), 1.5 μl of NEBuffer 2.1:10× concentration (BioLabs), and 3.0 μl of water.

[0071] 1. Place the reaction mixture in a 0.2 mL PCR tube and react at 60°C for 5 hours. 2. MetaPhor™ Agarose (Lonza) at a concentration of 3% is used for electrophoresis. 3. Use 1% TBE as the buffer and perform electrophoresis at 100 V for 45 to 50 minutes.

[0072] Figure 11 shows the bands obtained by electrophoresis. Sample 6 shows the resistant type band pattern e. Cattle from samples in which band pattern e was detected are resistant to ELV.

[0073] (Test Example 3: Detection of BLV by ddPCR) Blood samples taken from three cows (samples 1 to 3) on a farm in Miyazaki Prefecture were measured using quantitative PCR (qPCR, Test A), ddPCR using primers and probes targeting the env gene of Test Example 1 (Test B), ddPCR using primers and probes targeting the BLV pol gene (Test C), and multiplex ddPCR using primers and probes targeting the env gene of Test Example 1 in combination with primers and probes targeting the pol gene (Test D).

[0074] For qPCR in Test A, a bovine leukemia virus detection kit (RC201A, Takara Bio Inc.) was used according to the instructions attached to the kit. ddPCR in Test B was performed in the same manner as the ddPCR in Test Example 1. Test C was performed in the same manner as the ddPCR in Test Example 1, except that the forward primer, reverse primer, and probe were replaced with a forward primer, reverse primer, and probe targeting the pol gene. The nucleotide sequences of the forward primer, reverse primer, and probe targeting the pol gene are shown in SEQ ID NOs: 9, 10, and 11, respectively. The probe targeting the pol gene was also labeled with FAM.

[0075] In Test D, ddPCR was performed in the same manner as in Test Example 1, except that the PCR reaction solution (20 μl) consisted of 10 μl of 2×ddPCR Supermix, 0.5 μl of primer (env gene), 0.5 μl of primer (pol gene), 1 μl of primer (RPP30 gene), 2 μl of template, and 6 μl of water.

[0076] (result) In the case of qPCR in Test A, amplification was confirmed for Sample 1 and Sample 2 (A1 and A2 in Figure 12), but Sample 3 showed a high Ct value and an abnormally low signal (A3 in Figure 12). In ddPCR targeting the env gene in Test B, signals indicating amplification were detected for Sample 1 and Sample 3 (B1 and B3 in Figure 12), but not for Sample 2 (B2 in Figure 12). pol In ddPCR targeting the genes, signals indicating amplification were detected for Samples 2 and 3 (C2 and C3 in Figure 12), but not for Sample 1 (C1 in Figure 12). In ddPCR targeting the pol gene and env gene in Test D, signals indicating amplification were detected for all Samples 1 to 3 (D1, D2, and D3 in Figure 12).

[0077] The above-described embodiments are intended to explain the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not by the embodiments. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention. [Industrial Applicability]

[0078] The present invention is suitable for preventing infectious diseases in livestock. [Explanation of symbols]

[0079] 1 presentation unit, 2 output unit, 10 memory unit, 11 farm data, 12 presentation program, 20 RAM, 30 input device, 40 display device, 50 CPU, 60 bus, 100 quarantine support device

Claims

1. A presentation unit is provided which presents disease prevention measures based on data showing the infection amount of bovine infectious lymphoma virus in individual cattle and information about the farm where the individual cattle are raised, The information about the farm is The information includes at least one of whether or not cattle individuals can be culled or replaced, whether or not they can be kept in isolation, whether or not they can be kept in tandem, and whether or not newborn cattle individuals can be kept in isolation, The data showing the infectious dose is Proviral copy number quantified by droplet digital PCR, The primers used in the droplet digital PCR are A forward primer having a base sequence shown in SEQ ID NO: 1 and a reverse primer having a base sequence shown in SEQ ID NO: 2, Epidemic prevention support equipment.

2. The primers used in the droplet digital PCR are Further comprising a forward primer having a base sequence shown in SEQ ID NO: 9 and a reverse primer having a base sequence shown in SEQ ID NO: 10, The epidemic prevention support device according to claim 1.

3. The presentation unit The disease prevention measures are proposed based on the presence or absence of specific antibodies against the bovine infectious lymphoma virus in the blood of the cattle. The epidemic prevention support device according to claim 1 or 2.

4. The presentation unit and proposing disease prevention measures based on the susceptibility of the cattle individual to the bovine infectious lymphoma virus. The epidemic prevention support device according to any one of claims 1 to 3.

5. The presentation unit comparing the genetic sequence of the infectious bovine lymphoma virus possessed by the individual bovine animal with the genetic sequence of the infectious bovine lymphoma virus detected in other bovine animals, and presenting information for estimating the route by which the individual bovine animal was infected with the infectious bovine lymphoma virus; The epidemic prevention support device according to any one of claims 1 to 4.

6. A presentation step of presenting disease prevention measures based on data showing the infection dose of bovine infectious lymphoma virus in individual cattle and information about the farm where the individual cattle are raised, The information about the farm is The information includes at least one of whether or not cattle individuals can be culled or replaced, whether or not they can be kept in isolation, whether or not they can be kept in tandem, and whether or not newborn cattle individuals can be kept in isolation, The data showing the infectious dose is Proviral copy number quantified by droplet digital PCR, The primers used in the droplet digital PCR are A forward primer having a base sequence shown in SEQ ID NO: 1 and a reverse primer having a base sequence shown in SEQ ID NO: 2, Epidemic prevention support methods.

7. Computer, a presentation unit that presents disease prevention measures based on data showing the infection load of bovine infectious lymphoma virus in individual cattle and information about the farm where the individual cattle are raised; The information about the farm is The information includes at least one of whether or not cattle individuals can be culled or replaced, whether or not they can be kept in isolation, whether or not they can be kept in tandem, and whether or not newborn cattle individuals can be kept in isolation, The data showing the infectious dose is Proviral copy number quantified by droplet digital PCR, The primers used in the droplet digital PCR are A forward primer having a base sequence shown in SEQ ID NO: 1 and a reverse primer having a base sequence shown in SEQ ID NO: 2, program.

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