JP7821464B2 - Epidemic prevention support device, epidemic prevention support method and program - Google Patents

Description

The present invention relates to an epidemic prevention support device, an epidemic prevention support method, and a program.

Contagious livestock 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 significant problem for humanity in terms of ensuring a stable food supply. Early detection and early countermeasures for infectious diseases are essential to control the outbreak of infectious diseases in livestock.

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 disease prevention measures. Establishing comprehensive technology or systems that can support the implementation of effective disease prevention measures against EBL is an urgent issue.

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.

Japanese Patent Application Laid-Open No. 2014-199556

The device disclosed in Patent Document 1 uses a Markov chain model to calculate the probability of an animal infectious disease spreading spatially to other farms when it occurs in a certain area, and then identifies farms where epidemiological testing should be conducted based on the calculated infection probability. While the device presents risk information based on simulations, it cannot present disease prevention measures based on the actual infection status of livestock infectious diseases on farms. The disease prevention measures that can be implemented on farms may be limited by the farm's rearing environment.

The present invention was made in consideration 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.

An epidemic prevention support device according to a first aspect of the present invention comprises:
a presentation unit that presents 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
It includes 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 .

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,
This may also be the case.

Moreover, 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 .
This may also be the case.

Moreover, the presentation unit
and proposing disease prevention measures based on the susceptibility of the cattle individual to the bovine infectious lymphoma virus .
This may also be the case.

Moreover, 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 ;
This may also be the case.

A quarantine support method according to a second aspect of the present invention comprises:
a presentation step of presenting disease prevention measures based on data showing the infection load of bovine infectious lymphoma virus in cattle individuals and information on the farm where the cattle individuals 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
It includes 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 .

A program according to a third aspect of the present invention comprises:
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
It includes 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 .

This invention can support the implementation of effective prevention measures against livestock infectious diseases.

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. FIG. 10 is a diagram illustrating farm data. FIG. 10 is a diagram illustrating an example of data to be analyzed. FIG. 2 is a diagram showing epidemic prevention measures (tasks) presented by the epidemic prevention support device of FIG. 1. FIG. 2 is a flowchart showing an epidemic prevention measure presentation process performed by the epidemic prevention support device of FIG. 1 . FIG. 2 is a flowchart showing a process for selecting quarantine measures for high-risk cattle by the quarantine support device of FIG. 1. FIG. 2 is a flowchart showing an information presentation process performed by the epidemic prevention support device of FIG. 1 . FIG. 10 is a diagram showing the results of examining the annealing temperature in Test Example 1. FIG. 1 shows the results of simultaneous measurement of the env gene of BLV provirus and the bovine RPP30 gene in Test Example 1. FIG. 1 is a diagram 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. FIG. 1 shows bands obtained by electrophoresis in Test Example 2. This 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.

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 terms "have," "include," and "contain" also include the meanings of "consisting of" and "consisting of."

(Embodiment)
The epidemic prevention support device 100 according to this embodiment will be described. The epidemic prevention support device 100 is an information processing device useful for planning feasible epidemic 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.

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 animal to another. Livestock infectious diseases include rinderpest, bovine pleuropneumonia, foot-and-mouth disease, epidemic encephalitis, rabies, vesicular stomatitis, Rift Valley fever, anthrax, hemorrhagic septicaemia, 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, and 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 nosemiosis.

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 also experience reduced milk production and immunity. Currently, the only way to prevent the spread of infection is to identify BLV-infected cattle and cull or isolate them.

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 via a bus 60.

The memory unit 10 includes non-volatile storage media such as a ROM (Read Only Memory), HDD (Hard Disk Drive), and flash memory. The memory unit 10 stores various data and software programs, as well as farm data 11 and a presentation program 12.

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 they can be kept in isolation, whether they can be kept in tandem, and whether newborn calves can be kept in isolation. The farm data 11 illustrated in Figure 2 is a table in which, for each of the multiple farms F1, F2, and F3, "Yes" is assigned for cattle culling or replacement, isolation, tandem, and newborn calf isolation, and "No" is assigned for those options. Isolation refers to keeping one or more cattle in an independent barn (isolation barn) isolated from the rest of the herd. When isolation is possible, for example, by moving infected cattle from a herd kept in a free-stall barn to an isolation barn, the transmission of BLV from infected to uninfected 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 by fixing or tying them up. 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.

Returning to FIG. 1(A), RAM 20 functions as the main memory of CPU 50, and when CPU 50 executes presentation program 12, presentation program 12 is expanded in RAM 20. Data input via input device 30 is temporarily stored in RAM 20.

The input device 30 is hardware that allows a user to input data into the disease prevention support device 100. 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 using known techniques, such as real-time PCR or ddPCR. PCR can be performed using primers that target the BLV env gene or pol gene, for example. Alternatively, the provirus copy number can be quantified using multiplexed PCR, which uses multiple primer pairs to amplify multiple gene regions.

Data input via the input device 30 includes farm data 11, the number of provirus copies detected 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. The data input via the input device 30 is stored in the memory unit 10.

Data indicating the presence or absence of BLV-specific antibodies in the blood is, for example, data indicating whether specific antibodies were detected (positive) or not (negative) using the Enzyme-Linked Immunosorbent Assay (ELISA) method. 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.

Data regarding 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 using PCR-RFLP (Restriction Fragment Length Polymorphism). Using PCR-RFLP, the presence or absence of susceptibility or resistance to BLV can be determined, for example, depending on the MHC class II DRB3 allele type. The MHC class II DRB3 allele type can be determined using the restriction enzymes Rsa I, Hae III, and BstY I.

The BLV gene sequence data is obtained using a known sequence analysis method. The gene sequence data for multiple BLVs detected from multiple cattle may be stored in advance in the memory unit 10. In this case, the gene sequence data is stored in the memory unit 10 as a database that can be compared with the gene sequence data input as the subject of analysis.

Figure 3 shows an example of the data to be analyzed that was input via the input device 30. The data corresponds to IDs (1-32) assigned to each individual cow raised on the farm, along with 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.

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 memory unit 10 into the RAM 20 and executes the presentation program 12 to realize the functions described below.

Returning to Figure 1, Figure 1(B) 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.

The presentation unit 1 presents 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 presentation unit 1 presents disease prevention measures that can be implemented on the farm by referencing the farm data 11 according to the provirus copy number. Furthermore, the presentation unit 1 presents disease prevention measures based on the presence or absence of specific antibodies and susceptibility to BLV in addition to the provirus copy number.

Figure 4 shows the disease prevention measures (actions) presented by the presentation 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, the presentation unit 1 presents 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), the presentation unit 1 presents 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 with which BLV replicates in the body, such as the presence or absence of susceptibility to BLV as determined by the DRB3 allele type described above.

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 on 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.

The presentation unit 1 compares the genetic sequence of the BLV possessed by the cow with the genetic sequence of BLV detected in other cows, and presents information for estimating the route of BLV infection in the cow. When comparing the genetic sequences, the presentation unit 1 determines, for example, by phylogenetic tree analysis, whether the BLV types match or whether the BLVs are closely related. Phylogenetic tree analysis can be performed by known methods. The presentation 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.

The quarantine countermeasure presentation process performed by the quarantine support device 100 will be explained in further 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 quarantine countermeasure presentation process begins.

When a user inputs the data shown in Figure 3 into the epidemic prevention support device 100 via the input device 30 and issues an instruction to start the epidemic prevention measure presentation process, the epidemic prevention support device 100 starts the epidemic prevention measure presentation process. As shown in Figure 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 antibodies in the acquired cow data are positive or not (step S4). If the specific antibodies are not positive, i.e., negative (step S4; No), the presentation unit 1 determines whether the number of provirus copies is one or more (step S5). If the number of copies of the provirus is not 1 or more, i.e., if the number of copies is 0 (Step S5; No), the presentation unit 1 associates N (= ID) with #1 and stores it in the memory unit 10 (Step S6). If the number of copies of the provirus is 1 or more (Step S5; Yes), the presentation unit 1 associates N with #2 and stores it in the memory unit 10 (Step S7).

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).

If the provirus copy number is 500 or more (Step S10; No), the presentation unit 1 performs the high-risk cattle quarantine measure selection process (Step S12). Here, the high-risk cattle quarantine measure selection process will be explained with reference to Figure 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 high-risk cattle quarantine measure selection process ends. On the other hand, if there is no susceptibility, i.e., resistance (Step S24; No), the presentation unit 1 associates N with #10 and stores it in the memory unit 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 associates N with #11 and stores it in the memory unit 10 (Step S27), and ends the process of selecting quarantine measures for high-risk cattle.

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 associates N with #6 and stores it in the memory unit 10 (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 bridging rearing is possible (Step S30). If bridging rearing is possible (Step S30; Yes), the presentation unit 1 associates N with #7 and stores it in the memory unit 10 (Step S31), and ends the high-risk cattle quarantine measure selection process. If bridging rearing is not possible (Step S30; No), the presentation unit 1 refers to the farm data 11 and determines whether 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 associates N with #8 and stores it in the memory unit 10 (Step S33), and the process of selecting quarantine measures for high-risk cattle ends.

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 associates N with #12 and stores it in the memory unit 10 (Step S35), and ends the process for selecting quarantine measures for high-risk cattle. If there is no data indicating susceptibility (Step S34; No), the presentation unit 1 associates N with #11 and stores it in the memory unit 10 (Step S27), and ends the process for selecting quarantine measures for high-risk cattle.

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 references the memory unit 10 and displays N and the quarantine measures (#1-12) 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.

Estimating the infection route is useful for disease prevention measures. In accordance with the user's instructions, the presentation unit 1 executes an information presentation process that presents information for estimating the infection route. The information presentation process will be explained with reference to Figure 7. Note that the memory unit 10 has pre-stored genetic sequence data of BLV carried by multiple infected cattle raised on farm F.

The presentation unit 1 waits until a command 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.

The epidemic prevention support device 100 according to this embodiment presents epidemic prevention measures based on the BLV provirus copy number 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.

Farm data 11 may include at least one of the following: whether cattle can be culled or replaced, whether they can be kept in isolation, whether they can be kept in tethered housing, 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 cattle culling or replacement → isolation housing → tethered housing → isolation housing of newborn calves, thereby presenting quarantine measures that are feasible for each farm and have the highest preventive effectiveness. Note that farm data 11 is not limited to data indicating whether cattle can be culled or replaced, whether they can be kept in isolation, whether they can be kept in tethered housing, 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 present.

Note that the quarantine measures shown in Figure 4 are merely examples and are not limiting. The presentation unit 1 may present quarantine measures according to the amount of provirus. For example, the presentation unit 1 may present 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 presentation unit 1 may present a quarantine measure of not allowing infected cattle whose provirus copy number exceeds a threshold to produce calves.

Data indicating the amount of infection includes the number of provirus copies quantified by ddPCR or other methods. Because ddPCR can quantify the amount of BLV with high accuracy, the epidemic prevention support device 100 can present feasible and highly effective epidemic prevention measures based on highly accurate information related to infection risk.

The presentation unit 1 presents 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 presentation 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.

The display unit 1 suggests disease prevention measures based on susceptibility to BLV. This allows the display unit 1 to suggest cattle that should be prioritized for culling, replacement, and retention, allowing users to implement efficient disease prevention measures.

The presentation unit 1 compares the genetic sequence of BLV carried by the cow with genetic sequences of BLV detected in other cows, and presents 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 presentation unit 1 may also present epidemiological associations between farms by combining data on the movement history of cattle between farms with the results of phylogenetic tree analysis.

The presentation unit 1 may also determine whether the specific antibody is positive after determining whether the provirus copy number is one or more. If the provirus copy number is zero and the specific antibody is negative, the presentation unit 1 stores N in the memory unit 10 in association with #1; if the provirus copy number is one or more and the specific antibody is negative, the presentation unit 1 stores N in the memory unit 10 in association with #2. If the provirus copy number is zero and the specific antibody is positive, the presentation unit 1 stores N in the memory unit 10 in association with #3; if the provirus copy number is one or more and the specific antibody is positive, the presentation unit 1 executes step S10 and subsequent steps.

The epidemic prevention support device 100 may also 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 enter an ID that can identify an individual cow, thereby viewing the epidemic prevention measures associated with the ID stored in the memory unit 10. There may be multiple farms, and in response to access using the farm's unique identification information, the epidemic prevention support device 100 may transmit to the producer's terminal via the Internet information such information as the test results for the cows 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 cows relative to the number of cows raised on the farm), a ranking of transmission risk based on the number of provirus copies in infected cows, test history, and the IDs of untested cows.

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-ROM (Compact Disc Read Only Memory), DVD (Digital Versatile Disc), Magneto-Optical Disc, USB (Universal Serial Bus) memory, memory card, and HDD. 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 can be stored in a storage device owned by another server on the Internet, and the presentation program 12 and various software programs can be downloaded from the server.

The present invention will be explained in more detail using the following examples, but the present invention is not limited to these examples.

(Test Example 1: Quantification of BLV provirus by ddPCR)
Blood samples were collected from 139 cattle 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).

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 Inc.) and Cycleave PCR™ Reaction Mix SP (Takara Bio Inc.) on an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems).

To detect the BLV provirus using ddPCR, primers and probes were designed using the BLV env gene as the target 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.

The PCR reaction mixture (20 μl) consisted of 10 μl of 2x ddPCR Supermix, 1 μl of primer (env gene), 1 μl of primer (RPP30 gene), 2 μl of template, and 6 μl of water. Thermal recycling 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.

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

To analyze the correlation between real-time PCR measurements and ddPCR measurements, Spearman's rank correlation coefficient was used, with a significance level of 5%.

(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 a correlation with real-time PCR was analyzed. A significant correlation was confirmed between the two, as shown in Figure 10.

(Test Example 2: Discrimination of DRB3 allele types 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. Allow to 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 to stand 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, and 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 with a 200 μl pipette and leave the lid open to air dry.
13. Add 100 μl of sterilized water and leave to stand at 65°C for 1 hour or at 4°C overnight.
14. Measure DNA concentration with NanoDrop 8000.

Next, the BoLA-DRB3 exon 2 region is amplified by PCR. The following reaction mixture is prepared using the TaKaRa Ex Taq Hs kit (Takara Bio Inc.). The base sequences of primer (HL030) and primer (HL032) are shown in SEQ ID NOs: 7 and 8, respectively. The reaction mixture consists of 14.5 μl of water, 2 μl of 10x buffer, 2 μl of dNTP, 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).

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

The PCR product is digested with the restriction enzyme BstY I (BioLabs) as follows, and the DRB3 allele type is determined based on the size of the digested fragments confirmed by electrophoresis. The reaction solution consists of 10 μl of PCR product, 0.5 μl of BstY I (10,000 U/ml), 1.5 μl of NEBuffer 2.1:10x concentration (BioLabs), and 3.0 μl of water.

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

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

(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 according to Test Example 1 (Test B), ddPCR using primers and probes targeting the BLV pol gene (Test C), and multiplex ddPCR using a combination of primers and probes targeting the env gene according to Test Example 1 and primers and probes targeting the pol gene (Test D).

For qPCR in Test A, a bovine leukemia virus detection kit (RC201A, Takara Bio Inc.) was used according to the instructions provided with the kit. ddPCR in Test B was performed in the same manner as the ddPCR in Test Example 1. For Test C, the forward primer, reverse primer, and probe were replaced with a forward primer, reverse primer, and probe targeting the pol gene, and ddPCR was performed in the same manner as the ddPCR in Test Example 1. 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.

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 2x 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.

(result)
In the case of qPCR in Test A, amplification was confirmed for Samples 1 and 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 Samples 1 and 3 (B1 and B3 in Figure 12), but not for Sample 2 (B2 in Figure 12). In ddPCR targeting the pol gene in Test C, 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 and env genes in Test D, signals indicating amplification were detected for all Samples 1 to 3 (D1, D2, and D3 in Figure 12).

The above-described embodiments are intended to explain the present invention and are not intended to limit the scope of the present invention. In other words, the scope of the present invention is defined by the claims, not the embodiments. Various modifications made within the scope of the claims and their equivalents are considered to be within the scope of the present invention.

The present invention is suitable for preventing infectious diseases in livestock.

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 (7)

a presentation unit that presents 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 equipment. 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 . 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 . 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 . 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 . a presentation step of presenting disease prevention measures based on data showing the infection load of bovine infectious lymphoma virus in cattle individuals and information on the farm where the cattle individuals 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. 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.