Ear Notch Samples Research Articles

Monitoring for Bovine Viral Diarrhea Virus in Austrian Red Deer (Cervus elaphus elaphus) by using Ear-notch Samples

During 2007-09, ear-notch samples from free-living (n=527) and farmed (n=237) Austrian red deer (Cervus elaphus elaphus) were tested for bovine viral diarrhea virus type 1 (BVDV-1) and type 2 (BVDV-2) by enzyme-linked immunosorbent assay and single-tube real-time reverse transcription PCR. Ear-notch samples were collected by applying modified ear tags from randomly selected hunter-harvested red deer and from individuals originating from deer holdings. All samples tested negative for BVDV-1 and BVDV-2. Results of this study show no evidence of persistently infected animals. They indicate further that BVDV is playing a minor role in free-living and farmed red deer in Austria. Ear-notch samples are an effective tool for in vivo and postmortem detection of BVDV in wildlife. This sample collection technique can be easily used in combination with tagging individual wild animals kept in captivity.

Testing of Cattle Ear Notch Samples Using a Bovine Viral Diarrhea Virus NS3 Antigen ELISA Kit for Detection of Persistently Infected Animals

Identification and elimination of persistently infected (PI) cattle is important to controlling bovine viral diarrhea virus (BVDV) in herds. Compared to many diagnostic laboratory techniques, ELISA (enzyme linked immunosorbent assay) is convenient due to the ability to test large number of samples rapidly and consistently. A virus can be detected by probing for a specific nonstructural antigen like the NS3 (p80 in former nomenclature) or a structural antigen like the glycoprotein Erns (E0 or gp48 in former nomenclatures). Ear notch samples can be obtained easily and quickly from cattle. Another important advantage of ear notch skin biopsy samples is that they contain a high concentration of BVDV antigen. Few publications can be found describing the detection of NS3 in ear notches from cattle. The objective of this study was to compare the performance of a current commercial ELISA which specifically detects the NS3 antigen on ear notches from cattle to other tests utilized in diagnostic laboratories.

Outbreak of Salmonella enterica serotype Newport in a beef cow-calf herd associated with exposure to bovine viral diarrhea virus

Severe disease and death in cows and calves affected 1 of 3 separate groups (A, B, and C) of cattle on a commercial cow-calf operation. Clinical illness consisting of severe watery and bloody diarrhea, dehydration, weakness, and death affected adult cows and calves in 1 group (group B). Salmonella enterica serotype Newport was recovered from tissues of cows and calves from group B. Despite supportive and antimicrobial treatment of cattle in group B, cow mortality rate attributable to salmonellosis in that group was 7.9% (32/407); calf mortality rate was 14.4% (52/361). None of the cows in Groups A or C died, and the calf mortality rate in those groups was low. Salmonella enterica serotype Newport was recovered from pooled fecal samples subsequently collected from each group of cows. Bovine viral diarrhea virus (BVDV) antigen was identified in an ear notch sample collected from a necropsied calf from group B. Subsequently, ear notch specimens from cattle in all 3 groups were tested for BVDV antigen. A significantly higher proportion of calves persistently infected with BVDV was identified in group B (8/295 [2.7%]), compared with the proportion in groups A and C combined (1/287 [0.3%]). Outbreaks of disease attributable to Salmonella Newport infection in beef cattle are unusual. Because of the immunosuppressive nature of BVDV, the possibility of animals persistently infected with BVDV within the herd should be considered during investigation of unusual outbreaks of infectious diseases.

Effects of ear-notch sample handling on reliability of antigen-capture enzyme-linked immunosorbent assay testing for bovine viral diarrhea virus.

Antigen-capture enzyme-linked immunosorbent assay (ACE) testing of skin samples obtained by ear-notching is commonly used to detect animals persistently infected (PI) with bovine viral diarrhea virus (BVDV). At times practitioners may not have access to phosphate buffered saline (PBS) solution for specimen storage as suggested by the ACE kit manufacturer. The first objective of this study was to observe the effects of storing ear-notch samples in varying amounts of PBS on ACE results. The second objective was to observe the effects of delayed time before the ear-notch sample was placed in PBS on the ACE results. Using virus isolation and immunohistochemistry, nine PI-positive and eight PI-negative calves were identified to obtain ear notch biopsies for the study. The amount of PBS used to store both positive and negative ear-notch samples was varied (1, 2, 4, 6, 8 and 10 mL) in the first trial. In the second trial, the addition of PBS to both positive and negative samples was delayed post-collection; phosphate buffered saline was added to ear-notch samples at 0, 1, 2, 4, 8, 12 and 24 hours post-collection. Following sampling and application of treatments, all samples were tested in duplicate for BVDV antigen following the guidelines of the ACE kit manufacturer. Although the S/P ratios were different among treatment groups in both studies (P<0.05), neither varying the amount of PBS added to the sample vial nor delaying the addition of PBS to the sample affected the outcome when testing positive or negative samples.

Evaluation of two commercial enzyme-linked immunosorbent assays for detection of bovine viral diarrhoea virus in serum and skin biopsies of cattle

AIM: To assess the ability of two commercial bovine viral diarrhoea (BVD) virus (BVDV) antigen-capture enzyme-linked immunosorbent assays (ELISAs) to detect virus in serum and skin biopsies. METHODS: Thirty cattle persistently infected (PI) with BVDV were identified using routine diagnostic laboratory testing. Additional ear-notch skin biopsies and blood samples were collected from these animals to confirm the diagnosis, and from 246 cohorts, to determine their BVDV status. Skin biopsies were soaked overnight in buffer and the eluate collected. All sera and eluate were tested using two commercially available ELISAs for detecting BVDV antigen, and a subsample of positive and negative sera was tested using a polymerase chain reaction (PCR) test. A study was also performed to ascertain the risk of cross-contamination occurring during the collection and processing of skin biopsies. RESULTS: Both serum and skin samples tested using either ELISA resulted in the detection of all cattle identified as PI and no non-infected cattle were incorrectly classified as infected using either method. Agreement between all assays (ELISAs, whether performed on serum or skin, and PCR) was 100%. No cross-contamination of skin samples between animals was evident using routine biopsy methods. CONCLUSIONS: Viraemic cattle infected with BVDV were accurately identified using either of the two commercial ELISAs evaluated on either serum or skin samples. CLINICAL RELEVANCE: Either skin biopsies or serum samples can be collected from cattle to determine their BVDV status. This should overcome problems in accurately identifying the infection status of young calves in which colostral antibodies might interfere with the antigen-capture ELISA.

Parameters of Ear Notch Samples for BVDV Testing

Infection with bovine viral diarrhea viruses (BVDV) is a major source of economic loss for the US cattle industry. Houe (2003) estimates that losses in areas where BVD is endemic range between $10 to 40 million per million calvings. Results of serology surveys in the US suggests that our losses are in the upper end of this range (Houe et al, 1995a; Houe et al, 1995b; Paisley et al, 1996). In 2004, 37.6 million calves were born in the US; thus, US losses by this estimate would be between $376 million to 1.5 billion for 2004. The 2005 calf crop was ~37.8 million, making the 2005 estimate very similar to that of 2004.
 While BVDV infections are well recognized as reproductive pathogens among dairy producers, producers are less aware that BVDV infections are also associated with increased respiratory disease, increased severity of secondary infections and decreased milk production. Persistently infected (PI) animals are the major vectors for spreading BVDV within and among herds. Based on studies of dairies of 100 cows or more, Joly et al (2005) estimated that the presence of one PI animal in a herd resulted in a loss of $1.93/cwt of milk sold. Studies estimate that 10-15% of US herds have at least one PI animal (Houe et al, 1995c; Wittum et al, 2001). In 2004, US dairies produced 170,805 million lb (77,638 kg) of milk. If 10% to 15% of dairy herds have at least one PI animal, then the cost to milk production is between $330 to 494 million per year.
 Control efforts in the US are geared towards identifying and eliminating Pis. Several tests based on detection of either antigen or viral RNA in blood, serum, bulk milk or skin biopsies are currently in use (Cornish et al, 2005; Fulton et al, 2006). Ear notch samples have become the tissue of choice for screening for PI animals because 1) they are easy to collect; 2) equipment requirements are minimal; 3) they are not affected by presence of passive antibodies; and 4) they can be used as the sample for a wide variety of tests including immunohistochemistry, real-time polymerase chain reaction and antigen-capture ELISA.
 While ear notches have become one of the samples of choice, there is little information available regarding sample size requirements and stability. Further, while pooling of ear notch samples has been proposed for reducing the cost of surveillance programs (Kennedy et al, 2006), the viral load available for detection from ear notch samples is largely undetermined. The purpose of this study was to establish working parameters for sample size, viral detection limit and sample storage conditions for real-time PCR and antigen-capture ELISA.

Assessment of a Bovine Viral Diarrhea Virus Antigen Capture ELISA and a Microtiter Virus Isolation ELISA Using Pooled Ear Notch and Serum Samples

To reduce the cost of whole herd screening for bovine viral diarrhea virus persistently infected animals, the sensitivity and specificity of an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a microtiter virus isolation ELISA using saline from ear notch samples or pooled serum was determined. Pooled saline from ear notch samples, assayed by AC-ELISA, gave a sensitivity and specificity of 98% and 94%, respectively, for pools containing 2 samples and 72% and 100%, respectively, for pools of 5. The sensitivity of pooled ear notch or serum samples for bovine viral diarrhea virus detection by microtiter virus isolation (sensitivity < 5%) or serum samples for detection by AC-ELISA (sensitivity < 15%) is too low to be used for whole herd screening. Pooling saline from ear notch samples from 2 animals tested by AC-ELISA, however, could provide a less expensive, reliable method for whole herd screening for bovine viral diarrhea virus.

Reverse Transcription-Polymerase Chain Reaction on Pooled Samples to Detect Bovine Viral Diarrhea Virus by using Fresh Ear-Notch—Sample Supernatants

Ear-notch samples from 3,599 yearling heifers were collected to detect persistently infected (PI) animals with suspect bovine viral diarrhea virus (BVDV). Individual immunohistochemistry (IHC), individual antigen-capture enzyme-linked immmunosorbent assay (AC-ELISA), and reverse transcription-polymerase chain reaction (RT-PCR) tests with pooled ear-notch supernatants were compared with samples from 3,016 heifers, whereas RT-PCR ear-notch pools and individual AC-ELISA tests were compared with samples from all 3,599 heifers. Four heifers were identified positive by both IHC and AC-ELISA, whereas the remaining heifers were identified negative by both tests. When supernatant from ear notches from 100 animals was randomly pooled and RT-PCR was accomplished on each pool, RT-PCR identified 2 pools that contained 1 positive AC-ELISA sample and 1 pool that contained 2 positive AC-ELISA samples. Further evaluation of the pooled RT-PCR ear-notch supernatant detected 100% (n = 36) samples spiked with supernatant from a single randomly selected positive AC-ELISA ear notch. Although follow-up confirmatory tests were not completed, all 3 methods correlated 100% in detecting suspect PI animals, with a kappa value of 1. The use of RT-PCR on pooled ear-notch supernatant could provide an initial, rapid, cost-effective method of screening cattle herds for BVDV PI animals. Subsequent serial testing with an AC-ELISA to evaluate individual samples included in the positive pool could minimize the length of time other animals are exposed to the virus.

Preliminary evaluation of the effect of vaccination with modified live bovine viral diarrhea virus (BVDV) on detection of BVDV antigen in skin biopsies using immunohistochemical methods

Immunohistochemical (IHC) testing of skin biopsies (ear notch samples) is a method to identify cattle persistently infected (PI) with bovine diarrhoea virus (BDV). Skin biopsies were taken from 90 calves upon arrival (day 0) at a feedlot, and on days 14, 28 and 42 following arrival. Calves were vaccinated with modified live virus (MLV) BDV vaccine on days 1 and 14 following arrival. Additional samples were obtained from 50 of the calves between days 1 and 10 when they were treated for bovine respiratory disease. Immunohistochemical testing for BDV antigen was performed on all samples. One calf was positive for BDV antigen by IHC techniques on 5 skin biopsies. This calf was also positive by virus isolation (VI) on the buffy coat of a blood sample obtained on day 42 following arrival. All samples from the remaining calves were negative when tested with the IHC method. Other tests, VI and polymerase chain reaction, used to identify PI cattle may give positive results in cattle recently vaccinated with MLV BDV vaccine. This study suggests that IHC testing of skin biopsies may be advantageous over other tests to identify PI cattle recently vaccinated with MLV BVDV.