WO2011084619A2 - Élimination d'infection pathogène dans des populations d'animaux d'élevage - Google Patents

Élimination d'infection pathogène dans des populations d'animaux d'élevage Download PDF

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WO2011084619A2
WO2011084619A2 PCT/US2010/060813 US2010060813W WO2011084619A2 WO 2011084619 A2 WO2011084619 A2 WO 2011084619A2 US 2010060813 W US2010060813 W US 2010060813W WO 2011084619 A2 WO2011084619 A2 WO 2011084619A2
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pathogen
seq
madv
assay
pcr
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PCT/US2010/060813
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WO2011084619A3 (fr
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Mark Stahl
John Stahl
Richard Stahl
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Mark Stahl
John Stahl
Richard Stahl
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Priority claimed from US12/856,434 external-priority patent/US10081843B2/en
Application filed by Mark Stahl, John Stahl, Richard Stahl filed Critical Mark Stahl
Priority to EP10842612.3A priority Critical patent/EP2513338A4/fr
Publication of WO2011084619A2 publication Critical patent/WO2011084619A2/fr
Publication of WO2011084619A3 publication Critical patent/WO2011084619A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14311—Parvovirus, e.g. minute virus of mice
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14311—Parvovirus, e.g. minute virus of mice
    • C12N2750/14321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention provides a method to identify and remove pathogen infected animals from a group/herd to prevent the spread of infection and preserve the health of the animals.
  • PCR screening utilizing primers appropriate to the pathogen, of both the animals and their environs unambiguously identifies active infections.
  • antibiotics are administered to the animals on an intermittent or continuing basis.
  • the presence of the antibiotics or their by-products in consumable food products has raised concern about their long- term effect on human and animal health.
  • Immunization against some pathogens is another possible approach, but vaccines for many animal diseases are either not available or are not cost effective. Yet, for other pathogenic organisms no antibiotic or vaccine treatment is available.
  • Early detection of the infection and elimination/removal of the infected animals is the only method that can be used.
  • serologic detection methods vary in their sensitivity especially during the early days of infection and may only detect an infection after the animal has started to make antibodies to the pathogen and may, itself, already be infectious.
  • mink Aleutian Disease Virus This virus was first described in 1956. All mink Aleutian Disease Viruses are single stranded DNA viruses of the parvovirus family. There are many strains of the virus, but only one known non-pathogenic strain (strain G) while the others are typically fatal. The pathogenic viral strains are absolutely devastating to mink farmers spreading quickly through mink colonies and contaminating the farm site through contact with the mink and their urine and feces. These viruses typically elicit a hyperimmune response in the mink with lethality arising from macro immuno-antigen complexes. The hypergammaglobulinemia condition inflames circulatory filtering organs such as the kidneys (glomerulonephropathy), spleen, and liver causing failure of these organs and death from the complications.
  • mADV mink Aleutian Disease Virus
  • Mink Virus Enteritis MVE
  • Aleutian Mink Virus strain G
  • the patent basically deals with HIV identification and possible treatment methods are suggested for clinical treatment of infected patients. No direct application to infection with the deadly form of the Aleutian mink virus is discussed. Elford et al. In U.S. Patent No. 6,248,782 teach that polyhydroxy benzoic acid derivatives are useful in the treatment of diseases caused by retroviruses as well as in the treatment of diseases caused by DNA parvoviruses. No specific example of treatment for mink Aleutian disease is given. As far as is known, none of the above suggested approaches to containing a fatal mink Aleutian disease outbreak has been successfully employed.
  • inventive methods disclosed in this patent document are exemplified by the detection and eradication of pathogenic mink Aleutian disease virus from a farmed mammalian herd.
  • Figure 1 shows the number of mink deaths per week on a Pennsylvania farm infected with Aleutian mink disease for the years 2006 through December 2008.
  • Figure 2 is a photograph of a typical electrophoresis gel showing the locations of the GAPDH and mADV marker amplicons.
  • Figure 3 is the contiguous partial sequence corresponding to the Stahl mADV strain starting at approximately 272bp and ending at approximately 4440bp of the G strain (SEQ ID NO: 17).
  • Figure 4 shows the DNA sequence of the ADV G-strain (SEQ ID NO: 18) alongside the contiguous partial DNA sequence of the Stahl mADV strain (SEQ ID NO: 19) so far determined.
  • the alignment was obtained using Clustal W alignment utility located at
  • Figure 5 A is the amino acid sequence (SEQ ID NO: 20) of one protein specified by the Stahl mADV that does not include the hypervariable region. This protein is found at the same region of the genome as a protein found in the G strain.
  • Figure 5B is the amino acid sequence (SEQ ID NO: 21) of a second protein specified by the
  • Stahl mADV that does include the hypervariable region. This protein is found at the same region of the genome as a protein found in the G strain.
  • Figure 6 is a comparison of the partial amino acid sequences of several known mink
  • Aleutian disease viruses aligned (SEQ ID NOS 22-31, respectively, in order of appearance).
  • the hypervariable region is boxed (boxed sequence in StahlXl disclosed as SEQ ID NO: 32) .
  • Figure 7A is an outline of the screening method of the invention indicating the type of test applied at each stage and the disposition of animals that tested positive and negative.
  • Figure 7B is an outline of an embodiment of the screening method of the invention in which PCR mADV screening, but not antibody detection, in blood is used.
  • Figure 7C is an outline of a preferred embodiment of the screening method of the invention in which the herd is retested by PCR screening during the period roughly from December to February.
  • Figure 7D is an outline of a preferred embodiment of the screening method of the invention in which additional PCR testing of fecal material is performed at the time of whelping.
  • Figure 7E is an outline of a possible method to identify and place non-permissive animals into a breeding herd.
  • Figure 8A is a photograph of an electrophoresis gel showing the result of a composite placental manure PCR identification of mADV infection.
  • Figure 8B is a photograph of an electrophoresis gel showing on the left the result of PCR screening of the four females from the composite placental manure sample of Figure 8 A. None are mADV positive. The offspring of three of the four females were all mADV negative.
  • On the right of Figure 8B is the result of PCR screening of the 7 offspring of the fourth female from the composite placental manure sample of Figure 8 A. PCR identified three of the seven offspring as mADV negative while four of the seven offspring were PCR positive for mADV.
  • Figure 9 shows the number of mink deaths per week on a Pennsylvania farm infected with Aleutian mink disease for the years 2006 through September 2009.
  • IAT immunoagglutination test
  • CIEP CIEP sensitivity
  • CIEP a false negative window exists for at least one week post-infection, and CIEP will not determine if the virus was eliminated from the host.
  • the best results for CIEP were determined 1 year post test. ⁇ Cho, Greenfield, J Clinical Microbiology, Jan [1978] pp 18-22). If time and resources are available, post exposure antibodies can be detected at a fairly early stage using an ELISA assay (enzyme linked immunosorbent assay). Under farm conditions where a large number of animals (hundreds to thousands) need to be screened, a LFIA strip (lateral flow immunoassay) may be used in place of ELISA. Finally, ELISA is consistently more sensitive than CIEP (95%> vs.
  • PCR Polymerase Chain Reaction
  • the nucleotide sequence of the non-pathogenic Aleutian mink virus G strain was examined. This sequence had been published by Bloom.
  • the nucleotide sequence was obtained from PubMed.com (NCBI Reference Sequence NC 001662.1).
  • primers to a universal target, mink glyceraldehyde 3-phosphate dehydrogenase (mGAPDH) were developed.
  • the mGAPDH nucleotide partial sequence ⁇ Gram-Nielsen, et al.) was obtained from PubMed.com (GenBank: AF076283.1).
  • Multiplex PCR utilizes more than one set of PCR primers in the same reaction to allow
  • one pair of the multiplex PCR primers is used to detect the presence of the target in question while the other primer pair acts as an internal control to a universal target and assures that the quality of DNA extracted and PCR condition/technique is successfully implemented.
  • Multiplex PCR was used for all testing for the mADV.
  • the sequence of the hypervariable region was obtained. It was quickly realized that the mADV strain on the inventors' farm did not correspond to any strain in the published literature and was, therefore, a novel strain.
  • the sequence of the Stahl mADV genome was determined as indicated below in Section B (2). Subsequent to the initial identification of the first primer pairs, portions of the G strain sequence were entered into PrimerQuest and possible primers suggested. Selection of several primer pairs were made based on a judgment of what might work. Other primer pairs were tried over a course of about ten months in order to both identify the best primers to use to detect mADV and to identify primers having a sufficient coverage over the genome in order to sequence the entire virus genome.
  • primer selection is still an art and not an exact science and much trial and error was involved in determining useful primers. Some of the primer pairs worked while others did not, possibly due to mutual inhibition or to the inability of a particular region to anneal well. Primer pairs were suggested by PrimerQuest based on "relative abilities" to work as a primer based on the input sequence (or partial sequence). The remaining portions of the G-sequence were entered this way to find primers in the remaining untried regions.
  • the oligonucleotide primers themselves were obtained from Integrated DNA Technologies.
  • the DNA extraction conditions for PCR utilized by the inventors are set forth in Appendix "A”.
  • the PCR reaction conditions utilized by the inventors are set forth in Appendix "B”.
  • Table 1 lists several of the primer pairs generated, tested, and used in mapping and diagnostic screening based on the G-strain sequence. Those with amplicon sizes listed indicate that the pair worked well. As shown below, five of the primer pairs that were tried and expected to work have zero size indicating the pair did not work. The start and end positions numbers referenced correspond to the G strain sequence positions.
  • oligonucleotide sequences of some of the above primers used include:
  • V4 5' - ACA CGT GTC TTG GAG CAC TTC TCT - 3' (reverse) (SEQ ID NO: 6)
  • the primer pair used for mGAPDH is:
  • FIG. 1 is a photograph of a typical electrophoresis get and shows that the GAPDH and mADV amplicons are well resolved and separated.
  • V5 primers spanned the hypervariable region of the mADV (Bloom, et al . This not only yielded an amplicon distinguishable from the mGAPDH amplicon, but also enables the strain typing of the viruses by subsequent sequencing of this amplicon from different viruses
  • the V5 primer pair represents the preferred enablement and is used routinely as the diagnostic screening tool of choice for mADV.
  • reverse complement sequences of the above forward primers could also work as reverse primers (i.e. reverse complement of V5 forward equals V4 reverse).
  • reverse complement sequences of the above reverse primers could also work as forward primers (i.e.
  • any nested primers being a subset of the target region of the described primers, are included in the scope of this disclosure as are other primer pairs that overlap or are immediately adjacent to the primers described in detail above.
  • a novel mADV strain has been identified based on DNA sequences obtained from mADV amplicons produced from the PCR reactions using the above selected primers. Amplicons were sent to GeneWiz (GeneWiz, Inc., South Plainfield, NJ) for DNA sequencing. Overlapping DNA segments were assembled using DNA Baser software (dnabaser.com) to form a contiguous sequence.
  • Table 2 illustrates the relative alignment positions and sizes of the mADV amplicons used to sequence the mADV genome in relation to the G-sequence (vertical bars). Progression over time is indicated from top to bottom starting with V3/V2 and ending with V7/V7 ' . Hatched trellis regions indicate the part of the mADV DNA sequence obtained using the different primer pairs. Region 2.8kb (horizontal bars along the top of the table) indicates the relative hypervariable region (3096- 3134 bp). The assembled mADV contiguous region is depicted in the bottom row and was obtained from overlapping DNA sequences (273-4440 bp). It was assembled without any gaps by the use of the overlapping amplicons designed by proper primer pair placements. This is considered a partial sequence in relationship to the entire G-sequence since approximately the first 272bp at the 5 ' end and 361bp at the 3' end have not yet been identified.
  • the primer pairs that span the hypervariable region are V5-F/V5-R and V4b-F/V5b-R.
  • the contiguous partial sequence of the Stahl mADV strain is presented in Figure 3. While the standard procedure of starting the numbering sequence at "1" has been utilized in Figure 3, as noted above, the Stahl mADV sequence is a contiguous partial sequence starting about 272bp in from the start of the G strain sequence.
  • FIG. 4 A comparison of the nucleotide sequences of the G-strain and the Stahl strain is shown in Figure 4.
  • the alignment information shown in Figure 4 was generated using the Clustal W alignment utility located at http://www.ch.embnet.org/software/ClustalW.html.
  • the strain identifications, numbers, and primer designated sites have been added to the Clustal W comparison.
  • the primers that worked are shaded, while the primers that did not work are underlined.
  • the hypervariable region starting at 3096 (G-strain reference) is labeled and underlined.
  • the mADV contiguous sequence was BLAST searched against all other published sequences and no other identical match found (PubMed.com).
  • the mADV sequence shown in Figures 3 and 4 is the first time identification of the sequence of the highly infectious mADV virus has been determined.
  • any primer pair that spans the hypervariable region falling within the V5 primer pair including the nucleotide sequence of the hypervariable region disclosed in this patent document will generate a PCR amplicon specific to the Stahl mADV strain.
  • the hypervariable region specifies the strain type, use of such a primer pair that spans the hypervariable region with other ADV strains will permit an accurate strain typing that can be used to not only identify the strain but also to trace infections from place to place, herd to herd.
  • Figure 5 shows the amino acid sequence of two proteins predicted from the partial nucleotide sequence determined for mADV.
  • Figure 5A shows the amino acid sequence of one protein specified by the Stahl mADV that does not include the hypervariable region. This protein is found at the same region of the genome as a protein found in the G-strain.
  • Figure 5B shows the amino acid sequence of a second protein specified by the Stahl mADV that does include the hypervariable region. This protein is found at the same region of the genome as a protein found in the G-strain.
  • the sequences were generated using the ExPASy Proteomics Server, Swiss Institute of Bioinformatics (http://www.expasy.ch/tools/dna.html).
  • Figure 6 is a comparison over a limited span of the amino acid sequences of several mink viruses including the G-strain and the Stahl strain.
  • a nucleotide BLAST search was conducted using the Stahl strain nucleotide sequence as the query on PubMed.com (http://blast.ncbi.nlm.nih.gov).
  • Several similar DNA sequences obtained were selected for translation using ExPASy.
  • the comparative sequences span the hypervariable region (indicated by the box).
  • the amino acid sequence of the Stahl strain clearly differs from the others in several locations.
  • SNP's single nucleotide polymorphisms
  • Table 3 shows some SNP's identified by the DNA location and resulting change in coded amino acid.
  • Figure 7 shows in outline form the methodological sequence originally employed to identify and remove infected animals from the herd. Initially a visual examination of the animals is made to observe any animals showing clinical symptomology. Clinical signs such as lethargy, poor appetite,
  • LFIA can only detect antibodies after the 14-21 days it takes for the animal to mount a sufficient immune response.
  • a positive urine antibody test (using LFIA) on an asymptomatic animal indicates a prolonged and persistent viral infection and that sufficient renal damage (glomerulonephropathy) has already occurred from antibody/antigen complexes. Healthy animals will not excrete antibodies in urine unless the renal system has deteriorated. The antibody positive animals are removed from the herd, and, in the case of mink, are pelted.
  • An antibody negative urine animal is now a candidate for further antibody testing of its blood.
  • ELISA or LFIA may be used. Again, as noted above, LFIA is more conveniently used. LFIA testing of blood is a more sensitive test and does not rely on extensive renal damage having occurred. Blood is collected for both antibody testing and PCR testing at the same time according to the method described in Appendix "D". Whether the blood tests positive or negative for antibodies, the blood is still subjected to further PCR testing. In the case of an animal with antibody positive blood, it is possible that the animal has acquired a natural immunity to the virus and should be kept in the herd. If the blood PCR test indicates virus present in the antibody blood positive animal, the animal is removed from the herd.
  • the blood PCR test indicates no virus present in the antibody blood positive animal, the animal is kept in the herd and identified as antibody (+) virus (-). If the blood PCR tests positive for virus present in the antibody blood negative animal, the animal is removed from the herd. If the blood PCR test indicates no virus present in the antibody blood negative animal, the animal is kept in the herd and identified as antibody (-) virus (-). At this point in the selection process, both the antibody (+) virus (-) and the antibody (-) virus (-) animals are kept in the herd.
  • PCR mADV positive blood tests indicate an infected animal and indicate that the animal should be removed from the herd.
  • PCR mADV negative animals are kept in the herd.
  • the preferred protocol embodiment of the invention is outlined in Figure 7B.
  • the healthy animals are transferred to sanitized pens.
  • the animal is caught with Oxine soaked gloves (500 ppm), placed in a small carrier and dowsed repeatedly in a 200 ppm solution of Oxine.
  • Into this solution is also added a small amount of dish washing soap to aid as a surfactant for the aqueous Oxine solution to penetrate the highly hydrophobic under wool.
  • Oxine aids in the elimination of environmental virus on the mink. It has been discovered that it is possible to have a viral blood negative mink in a viral positive pen.
  • the 200 ppm Oxine solution was not found to have any effect on the eyes or mucus membranes of the mink and is an effective tool for killing the virus in the mink's coat. Only after undergoing this cleansing methodology was a mink placed into a freshly sanitized, quarantined, windward area of the ranch.
  • retesting of the animals using the preferred PCR protocol may be required to either confirm the absence of the virus in the herd or to remove any remaining infected animals. Based on the inventors' experience, it is believed that the optimum windows for testing are December during pelting, late February prior to breeding, and whelping season. PCR retesting according to the protocol set out in Figure 7C of the mink herd on the inventors' farm two months after the above described testing and selection process discovered that about 1.5% of the females and less than 1% of the males were still infected. In addition, the pens of these animals were resanitized and left dormant.
  • the viral elimination protocols outlined above substantially reduced the mink mortality for 2009. It should be noted that a variety of causes unrelated to mADV infection result in some level of mink mortality as is reflected in Figure 9 for 2009. However, it should also be appreciated that the viral elimination protocols and hygienic cleaning of the farm result overall in a much healthier herd.
  • Placental Manure Sampling The females of many mammalian animal species, including mink, soon after giving birth devour the discharged placenta. Malformed or dead offspring may also be consumed. The reason for this behavior is not well understood but may be linked to the need for hormones to reduce uterine bleeding (in mammals).
  • mink shortly after consumption of the placenta, the female mink passes a black, tarry, and shiny stool. Typically the stool is found in a far corner of the pen or even on the ground. If deposited relatively soon prior to discovery, the stool is easily sampled by inserting a small diameter tube a fixed distance into the medium to collect a sample size of approximately 35uL. This sample is placed in a labeled tube for submission for PCR. If the stool has been deposited for some time and the weather conditions are dry, a hard skin begins to form around the medium that must be broken for internal sampling.
  • mADV can be detected in animal manure as shown in Figure 8A.
  • the placental stool PCR mADV screening enables the removal from the herd of the affected dams and litters the day of whelp.
  • this method provides a non-invasive and non-tactile method of screening that does not disrupt the mink during this period with unnecessary handling.
  • the method minimizes the spread of the disease through contact and handling at the beginning of the spring and summer (the whelping season), the most contagious times of the year.
  • the animals are removed from the herd, and, in the case of mink, euthanized.
  • the litters adjacent to the infected litter are removed to pens on the leeward side of the ranch for quarantining and observation as an extra precaution. All empty pens are then cleaned and resanitized as taught previously.
  • analysis of the stool by PCR discovers infection in the offspring as well as the mother. Based on the discovery of pathogen detection by PCR in the placental manure of mink, detection of pathogen infection in the placental manure of other species in which the mother consumes the placenta may be accomplished by PCR analysis for a representative pathogen nucleotide sequence.
  • FIG. 8 A shows the results of PCR mADV analysis of 9 different pooled placental manure samples. As shown, the mADV virus was found in one pooled sample. Thus the dilution factor of the sample is not a concern due to the high sensitivity of the PCR method. Higher pooling numbers are possible but the limits have not been explored as of yet.
  • Figure 8B shows the PCR results for all four females (on the left of the central ladder column) and the seven offspring of one female (on the right of the central ladder column). All four females were found to be PCR mADV negative. Three of the four litters (18 offspring) were also PCR negative for mADV (not shown). The fourth PCR mADV negative female had a litter of 7 offspring in which 3 of the 7 were PCR mADV negative while 4 of the 7 were PCR mADV positive as shown on the right of the central ladder column of Figure 8B. Two very important discoveries come out of this data.
  • PCR testing of the placental manure picked up mADV infection in the offspring.
  • PCR mADV testing also revealed the presence of an otherwise healthy and PCR mADV negative female that carried the mADV virus and was capable of passing the virus on to her offspring.
  • Screening by the composite sampling method permits not only the identification of infected animals that were kept in the herd having passed the initial screening tests, but, most importantly, also permits the identification of carrier animals that need to be removed from the herd in order to eliminate all infection from the herd.
  • the PCR mADV negative female animal was "non-permissive"; that is, the virus is unable to infect the animal's cells even though virus particles remain sequestered in the animal.
  • the female apparently passed on her "non-permissive" genome to 3 of her offspring but not the other 4.
  • the relevant literature has taught that the vertical transmission of disease caused by mink disease virus was 100%. The results shown in Figure 8B clearly indicate otherwise.
  • Any PCR mADV negative kits would be segregated and used to establish a non-permissive herd. At this point it would be unknown whether the kits harbor a sequester virus and would transmit the virus to their offspring. Any remaining PCR mADV positive kits as well as the PCR mADV negative dam that is now known to harbor the virus would be pelted. Repetitive identification and segregation of non- permissive animals in subsequent generations should establish a gene line that breeds true for non- permissive animals.
  • saliva collection process for PCR can also be employed.
  • Saliva can be collected from mink by allowing them to bite upon a thin plastic tube or string or absorbent material such that sufficient saliva is collected. No handling of the animal is required which lessens the transmission of the disease and speeds collection. Typically this sampling is best achieved just prior to feeding time for the animals as they are very aggressive towards objects placed through the wire cage. The chewing process on the tube or string or other material is sufficient to deposit enough saliva for nucleic acid detection. Return visits may be required for animals that are not compliant.
  • sampling tube or string or other material may not touch the wire cage since environmental virus is likely to be included in the sample. Care must be taken at this point to ensure that no contamination results before the sample is safely placed in its labeled sampling container.
  • LFIA antibody testing
  • PCR testing the sampling lid is opened and the portion of tube or string or other material is cut off allowing it to fall into the container and then the lid is closed. Specific duties of each hand are practiced as described in Appendix "D".
  • the method of the present invention has been exemplified by application to the elimination of mADV from a mink herd.
  • the basic principles of screening using PCR detection of a pathogen's nucleic acid signature, with or without additional screening technologies such as antibody testing (ELISA or LFIA) to identify and remove infected animals from a herd has general applicability to a wide range of animals. The techniques may even be extended to populations of wild animals particularly through the PCR testing of manure.
  • kits may contain just the primers for mADV with the users supplying reference primers and laboratory facilities. More advanced kits may contain not only the mADV primers but also the GAPDH or other internal reference marker primers along with the remaining materials required to screen by PCR.
  • Samples received for detection of mink Aleutian Disease Virus (mADV) were processed using RNase/DNase free microcentrifuge tubes and sterile pipette tips containing aerosol filters. Samples collected consisted of 2mL microcentrifuge tubes containing either:
  • Total DNA from cotton swab and small tube samples was extracted and purified using Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc., Valencia, CA). The suggested manufacturer's protocol "Purification of Total DNA from Animal Blood or Cells (Spin-Column)" was performed. Minor changes were incorporated into the protocol for manure and placental manure samples. For samples containing more than one small tube, the Master Lysis Buffer volume was increased two fold, samples were applied to Spin Columns/Collection Tubes in 2 sequential loading applications (due to increased volume), 8000rpm spins for 1 minute were increased to 9000rpm for 3 minutes, and 13600rpm spin for 3 minutes increased to 6 minutes. Total DNA from cotton swab, small diameter tube, capillary tube, pipette tip, QIAcard (excised 2.5 sq mm), and saliva applicator samples was extracted using Epicentre QuickExtract DNA Extraction Solution (Epicentre).
  • PCR steps included initial denaturation (95°C for 2 minutes) followed by a 40 cycle loop of denaturation (95°C for 30 seconds), annealing (see table below), and extension (72°C for 1 minute), and then final extension (72°C for 5 minutes) with a hold at 4°C.
  • initial denaturation 95°C for 2 minutes
  • 40 cycle loop of denaturation 95°C for 30 seconds
  • extension 72°C for 1 minute
  • final extension 72°C for 5 minutes
  • PCR reactions were subjected to agarose electrophoresis. PCR products (amplicons) were visualized by UV fluorescence using GelRed Nucleic Acid Stain (Phenix Research Products, Candler, NC) incorporated in the agarose. The presence of the GAPDH amplicon (250bp) in the sample indicated that (cellular) DNA was extracted correctly and PCR performed properly. Appearance of the mADV amplicon (802bp for V5) indicated the presence of viral DNA in sample.
  • boxes are removable from their pens, they are immersed in a 3% NaOH solution as well as any other wooden- ware associated. These are then cleaned typically with a cleaning machine delivering 4 GPM @ 3000 PSI @ 190 degrees F. The outside surfaces of the box are done first finishing with the inside surfaces. Other parts are cleaned similarly whereupon the box with its parts are removed from the shed and immersed in a 500 ppm solution of Oxine (Bio-Cide
  • the next phase of cleaning addresses the wire pens and inside surfaces of the shed.
  • the pens are sprayed with a 3% NaOH solution with the optional addition of a foaming agent to enhance maximum contact to the extremely large surface area involved. While this is soaking, the inside roof and other areas are sprayed with a detergent [Complete Plus, (Cameo Chemical, Madison, WI)], again with the optional use of a foaming agent.
  • a detergent Complete Plus, (Cameo Chemical, Madison, WI)
  • the 3% NaOH solution is not recommended on surfaces that are aluminum such as shed roofs so the use of a detergent is used instead. Rinsing of the inner roof surface and other structural parts of the shed is preformed with the same machine initially before the wire pens are done working in a top to bottom fashion.
  • the pens are carefully rinsed in a manner that directs the spray to as many angles possible to minimize shadowed areas formed by the spraying action.
  • the pens are then sprayed with a 500 ppm solution of Oxine and allowed to air dry. Again the addition of a foaming agent enhances the contact time and completeness of the sanitizing solution.
  • the final step of preparing the shed is to broadcast CaO inside and outside of the shed by use of a garden pulled lawn broadcaster.
  • the CaO is applied at the rate of approximately six pounds per square yard.
  • the shed remains in this state until just prior to moving in PCR mADV negative animals.
  • a second application of 500 ppm Oxine is applied to the pens to ensure sanitation before use. Under all circumstances, strict ranch hygiene is absolutely essential for the successful implementation of eradication of the disease. Animal testing alone will not ensure elimination of a pathogen without adherence to the highest levels of biosecurity.
  • a technique of using Oxine soaked handling gloves is employed as to provide a sanitizing surface for any bodily fluids from the animals to be neutralized upon contact.
  • the gloves are soaked in a 500 ppm solution of Oxine until saturated and the handler first dawns a pair of latex gloves before the soaked catching gloves to protect his/her hands from the long term exposure to the Oxine solution.
  • the mink are carefully caught as to avoid contact of the rear feet with the Oxine laden gloves as it was discovered that Oxine will produce a false positive reaction on LFIA test strips when incorporated with the blood sample (personal communication).
  • the handler holds the animal horizontal with the rear feet to him/her and extended beyond the pen with the fore feet placed firmly on the top part of the pen while gently rolling the animal to the left side to raise the right rear foot upward.
  • the sampling person prepares to acquire the blood sample. Since a third hand is required, the mouth of the sampler may be used to hold the stem ends of the sterile cotton swabs while the right hand holds the clippers and is the only hand used to open and close sample containers. Reproducible non-cross contaminating sample acquisition is crucial at this stage. It is imperative that the sampler maintains a clean hand, usually the most dexterous one, and a sampling hand, one that is in repetitive physical contact with the animals. The two hands never exchange duties and maintain their respective operations.
  • the technique of blood collection is best preformed as follows. With the left hand, the sampler firmly grabs the elevated right rear foot of the mink such that the foot pad rests completely on the left thumb of the sampler. With the right hand, the sampler skillfully clips a toenail, preferably from the smallest, last digit, just above the quick line with a small pair of toenail clippers maintaining the grip with his left thumb and left fore finger of the left hand. Blood will flow momentarily or, if not, a second clipping may be required or a slight relaxation of the grip may allow the flow of blood to proceed. The sampler removes from his mouth a sterile cotton swab with his clean right hand and acquires first the sample for blood LFIA.
  • the stem of the swab is transferred to the released left hand, the pre-labeled sample container lid is opened with the thumb and fore finger of the right hand and the cotton head of the blood soaked swab is cut with the clippers still held in the right hand just above the cotton head. The lid is closed with the right thumb and fore finger. The stem of the swab is discarded with the left hand and is then used to re-grip the animal's right rear foot as before. Secondly, the sample for blood PCR is acquired in the same fashion excluding any contact with anything other than free flowing blood from the toenail to avoid environmental virus contamination. This process is repeated using the same hands in the same fashion as previously described.
  • the clippers Upon completion of acquiring samples from the animal, the clippers are wiped free of any blood with a paper towel using the left hand and exchanged with a second pair of clippers soaking in 500 ppm of Oxine.
  • This second pair is carefully dried with a clean portion of paper towel using the left hand but not allowing the sampling fingers to touch any part of the clipper's cutting surface. Layers of clean towel are maintained between the left fingers and cutting surface and the handles are held by the right hand.
  • the purpose of this drying action is to eliminate false positive LFIA that may arise with Oxine present in the blood sample.
  • the used towel is not discarded until used to remove blood from the next clipping action prior to immersion in Oxine. A fresh towel is only used for the pair of clippers immediately removed from the Oxine.
  • Blood collection can also be taken using 1.0 to 1.1 mm ID Na heparinized plastic capillary tubes commonly used in CIEP (counterimmunoelectrophoresis) testing, (Globe Scientific, Paramus, New Jersey).
  • the mink is similarly handled and hygiene observed as above only the use of a capillary tube instead of cotton swab acquires the sample.
  • volumes of samples can be accurately established due to the constant capillary diameter and length of tube filled. For instance a half-filled capillary tube is approximately 35 uL in volume.
  • the contents of the capillary tube are expelled by the use of a capillary bulb into a pre- labeled/bar coded sampling vial with a snap top or into a pre-labeled /bar coded 48 or 96 well plate suitable for extraction and/or PCR.
  • PCR technology allows testing of the environment for mADV presence. This is particularly important to eliminate the possibility of recontamination of the animals that are returned to the pens. Thus, environmental sampling is most useful after a cleaning procedure to determine the efficacy of the cleaning and sanitizing processes.
  • the method used is as follows. An area to be investigated is aggressively rubbed with a cotton swab that has been soaked in a Phosphate Buffered Saline (PBS, Boston BioProducts, Inc., Worcester, MA). The presoaking of the cotton swab aids in the acquisition and preservation of the sample.
  • PBS Phosphate Buffered Saline
  • the sampling area can include, but is not limited to, the wire cages, wooden boxes and their parts, inside of the housing roof surfaces, and the ground to name a few of the more obvious and worthwhile sites. As previously stated, the use of proper hygiene while manipulating the sample is always important.
  • the sample may be stored at 4 C° until the PCR process.

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Abstract

L'élevage d'animaux a toujours été sensible aux ravages d'infections pathogènes. Les grippes aviaires et maladies du bétail sont deux exemples qui ont des conséquences graves pour les animaux et affectent de façon indésirable les bénéfices économiques des éleveurs. La présente invention concerne une méthodologie d'essai et de réforme qui peut éliminer des pathogènes d'un troupeau infecté. La sensibilité de la PCR pour détecter de très faibles taux d'acide nucléique d'un pathogène infectieux est utilisée pour identifier des animaux infectés. De plus, il a été découvert que l'analyse PCR d'échantillons de fumier peut identifier précisément des animaux infectés et leur descendance pour les espèces qui consomment des résidus placentaires après la naissance. Le virus de la maladie aléoutienne du vison (mADV) est l'un de plusieurs parvovirus à ADN mortels qui peuvent atteindre rapidement des proportions épidémiques dans un troupeau de visons. Des amorces PCR ont été développées qui génèrent des amplicons pour détecter et identifier le mADV. De plus, une souche précédemment inconnue de mADV a été découverte, son génome séquencé, et sensiblement détaillée.
PCT/US2010/060813 2009-12-16 2010-12-16 Élimination d'infection pathogène dans des populations d'animaux d'élevage WO2011084619A2 (fr)

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US28688509P 2009-12-16 2009-12-16
US61/286,885 2009-12-16
US12/856,434 US10081843B2 (en) 2009-08-21 2010-08-13 Use of birthing manure to screen for pathogenic infection in farmed animals
US12/856,434 2010-08-13
US12/856,422 US20110086350A1 (en) 2009-08-21 2010-08-13 Elimination of pathogenic infection in farmed animal populations
US12/856,398 US10077481B2 (en) 2009-08-21 2010-08-13 Efficient multiple stage screening method to eliminate pathogenic infection in farmed animal populations
US12/856,398 2010-08-13
US12/856,422 2010-08-13

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EP0610444B1 (fr) * 1991-10-31 2003-01-29 ELFORD, Howard L. Utilisation d'un polyhydroxybenzamide ou de l'un de ses dérivés dans la fabrication d'un médicament destiné au traitement d'infections virales
EP1409730A2 (fr) * 2001-06-14 2004-04-21 University of Wales College of Medicine Procede de detection de virus, amorces correspondantes, et kit de criblage

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