Detection of Clinical and Subclinical Lumpy Skin Disease Using Ear Notch Testing and Skin Biopsies.

Lumpy skin disease (LSD) diagnosis is primarily based on clinical surveillance complemented by PCR of lesion crusts or nodule biopsies. Since LSD can be subclinical, the sensitivity of clinical surveillance could be lower than expected. Furthermore, real-time PCR for the detection of LSD viral DNA in blood samples from subclinical animals is only intermittently positive. Therefore, this study aimed to investigate an acceptable, easily applicable and more sensitive testing method for the detection of clinical and subclinical LSD. An animal experiment was conducted to investigate ear notches and biopsies from unaffected skin taken from the neck and dorsal back as alternatives to blood samples. It was concluded that for early LSD confirmation, normal skin biopsies and ear notches are less fit for purpose, as LSDV DNA is only detectable in these samples several days after it is detectable in blood samples. On the other hand, blood samples are less advisable for the detection of subclinical animals, while ear notches and biopsies were positive for LSD viral DNA in all subclinically infected animals by 16 days post infection. In conclusion, ear notches could be used for surveillance to detect subclinical animals after removing the clinical animals from a herd, to regain trade by substantiating the freedom of disease or to support research on LSDV transmission from subclinical animals.

The Occurrence of a Commercial Npro and Erns Double Mutant BVDV-1 Live-Vaccine Strain in Newborn Calves.

The major source for the spread of bovine viral diarrhea virus (BVDV) are in-utero infected, immunotolerant, persistently infected (PI) animals since they shed enormous amounts of viruses throughout their lives. During the sequence-based virus typing of diagnostic ear notch samples performed in the context of the obligatory German BVDV eradication program, the commercial Npro and Erns double mutant BVDV-1 live-vaccine strain KE-9 was detected in seven newborn calves; their mothers were immunized in the first trimester of gestation. Six calves either succumbed or were culled immediately, but the one remaining animal was closely monitored for six months. The viral RNA was detected in the skin sample taken in its first and fifth week of life, but the virus could not be isolated. Further skin biopsies that were taken at monthly intervals as well as every serum and urine sample, nasal, oral, and rectal swabs taken weekly tested BVDV negative. However, neutralizing titers against BVDV-1 remained at a consistently high level. To further control for virus shedding, a BVDV antibody and antigen negative calf was co-housed which remained negative throughout the study. The missing viremia, a lack of excretion of infectious virus and negative follow-up skin samples combined with consistently high antibody titers speak against the induction of the classical persistent infection by vaccination with recombinant KE-9 during gestation. We, therefore, suggest that the epidemiological impact of the RNA/antigen positivity for an extended period in the skin is very low. The detection of live-vaccine viruses in skin biopsies mainly represents a diagnostic issue in countries that implemented ear notch-based control programs; and KE9-specific RT-PCRs or sequence analysis can be used to identify these animals and avoid culling measures.

Primera evidencia del Virus de Diarrea Viral Bovina (VDVB) genotipo 2 en Colombia / First evidence of Bovine Viral Diarrhea Virus (BVDV) genotype 2 in Colombia

RESUMEN El Virus de la Diarrea Viral Bovina (VDVB) es un patógeno que afecta los hatos bovinos. El virus ha sido clasificado en dos biotipos (citopático y no citopático) y en 3 genotipos (1, 2 y 3) según su secuencia nucleotídica. El propósito de este estudio fue determinar la presencia del VDVB genotipo 2 (VDVB-2) en Colombia, mediante el estudio de cuatro zonas representativas de producción ganadera por medio de RT-PCR en muestras de suero y cartílago de oreja. Para ello, se recolectaron los sueros preparto de 379 vacas, de 274 terneros precalostrales (antes de que se les diera calostro), y de 145 terneros de 25 días post-nacimiento. Adicionalmente, se tomaron 181 biopsias de cartílago de oreja de estos terneros. Se realizó RT-PCR a todas las muestras para determinar la presencia o ausencia del VDVB. Aquellas muestras que resultaron positivas se evaluaron adicionalmente mediante dos métodos para determinar su genotipo: a) una nueva RT-PCR con primers específicos para el VDVB-2, y b) una PCR diferente con la que se obtuvo un producto de 296 pb, el cual se sometió a digestión enzimática. Los resultados mostraron que 17 (4,48%) muestras de suero preparto fueron positivas para Pestivirus, de las cuales 6 correspondieron al VDVB-2 (1,58%). Ninguna de las muestras de suero obtenidas de los terneros resultó positiva para el VDVB-2. Finalmente, 18 (9,9%) biopsias de cartílago de oreja fueron positivas al VDVB, 14 (7,7%) de las cuales resultaron positivas para el VDVB-2. El presente estudio es la primera evidencia documentada de la presencia del VDVB-2 en bovinos de Colombia.

ABSTRACT The bovine viral diarrhea virus (BVDV) is a pathogen that affects cattle. The virus has been classified into two biotypes (cytopathic and non-cytopathic) and three genotypes (1, 2 or 3) according to their nucleotide sequence. The objective of this study was to determine the presence of the BVDV genotype 2 (BVDV-2) in Colombia, through the study of four representative areas of cattle production by means of RT-PCR conducted on serum and ear notches. For this purpose, sera were collected from 379 prepartum cows, 274 calves born to these cows (before they were given colostrum), and 145 25-day- old calves. Additionally, 181 ear notches were taken from these calves. RT-PCR was performed on all samples to determine the presence or absence of BVDV. Tha samples that tested positive were further assessed by two methods to determine their genotype: a) a new RT-PCR with specific primers for BVDV-2, and b) a different PCR obtaining a product of 296 bp, which was further subjected to enzymatic digestion. The results showed that 17 (4.48%) prepartum sera samples were positive for Pestiviruses, from which 6 corresponded to BVDV-2 (1.58%). None of the sera obtained from the calves were positive for BVDV-2. Finally, 18 (9.9%) ear notches were positive for BVDV, 14 (7.7%) of which were positive for BVDV-2. The present study is the first documented evidence of the presence of the BVDV-2 in cattle from Colombia.

Factors associated with the bovine viral diarrhoea (BVD) status in cattle herds in Northwest Germany.

In Germany, all calves are tested for the presence of bovine viral diarrhoea/mucosal disease virus (BVDV) virus since January 1, 2011. The basis for this compulsory investigation is the BVDV Federal Regulation (BVDVV), which demands testing of calves before the age of six months and according to the new regulation of June 2016 within four weeks or before entering another stock. In 2012, a questionnaire was sent to 7250 Lower Saxony cattle farmers to identify potential factors associated with the presence of BVDV. Completed questionnaires were received from 2542 farms for further analysis. For BVD status determination of these farms, the diagnostic results of 425,911 ear notch samples of calves as part of the BVD eradication period from June 2010 to December 2013 were used. For the analysis of the completed questionnaires, a univariable analysis was performed by the chi-square or Wilcoxon test for each variable studied. In addition, a multivariable logistic model was performed. Four potential risk factors remained after a backward selection in the final logistic regression model: the dairy production compared to the suckling and other types of production, the herd size, the purchase of animals and the location in western region in comparison with the central and eastern regions. In summary, according to the results of this study, the farm with the highest probability of a BVDV infection in Lower Saxony is a large dairy farm that purchases cattle and is located in a cattle-dense region. When the complete eradication of the virus will be achieved, the results of the present study may help to conduct a risk-oriented monitoring programme.

Six Years (2011-2016) of Mandatory Nationwide Bovine Viral Diarrhea Control in Germany-A Success Story.

Bovine viral diarrhea (BVD) is one of the most important infectious diseases in cattle, causing major economic losses worldwide. Therefore, control programs have been implemented in several countries. In Germany, an obligatory nationwide eradication program has been in force since 2011. Its centerpiece is the detection of animals persistently infected (PI) with BVD virus, primarily based on the testing of ear tissue samples of all newborn calves for viral genome or antigen, and their removal from the cattle population. More than 48,000 PI animals have so far been detected and removed. Between the onset of the program and the end of 2016, the prevalence of these animals among all newborn calves decreased considerably, from 0.5% to less than 0.03%. The number of cattle holdings with PI animals likewise decreased from 3.44% in 2011 to only 0.16% in 2016. Since a large number of naïve, fully susceptible animals are now confronted with BVD virus, which is still present in the German cattle population, the challenge of the coming years will be the identification of remaining PI animals as quickly and efficiently as possible, and the efficient protection of BVD-free farms from reinfection.

A newly developed BVDV-1 RT-qPCR Taqman assay based on Italian isolates: evaluation as a diagnostic tool.

A single-step TaqMan® RT-qPCR was developed for the detection of bovine viral diarrhea virus type 1 (BVDV-1), an important pathogen of cattle worldwide. The assay was based on conserved 5'UTR sequences of Italian BVDV-1 isolates. In order to establish a diagnostic protocol which simplifies sample collection and processing, the assay was tested on a variety of biological specimens collected from persistently infected calves. The samples analyzed included PBMCs, plasma, dry blood, ear notch and hair bulb. Time and costs required for the analysis of each type of specimen were compared. The RT-qPCR, whose lower limit of detection was 100 copies of viral RNA (1 TCID50), correctly identified all PI animals, irrespective of the type of specimen. The highest copy numbers were obtained from the RNAs extracted from PBMCs, ear notches and hair bulbs. Hair bulb-supernatants directly used as a template allowed identification of all PI animals. In conclusion, based on time and cost evaluation, the most effective and efficient protocol was the one based on the direct analysis of hair bulb-supernatants, avoiding the RNA extraction step.

Using the mouse grimace scale to assess pain associated with routine ear notching and the effect of analgesia in laboratory mice.

Social housing is recommended where possible for laboratory mice. In order to achieve this, mice must be individually identifiable. Although, various methods are available, permanent identification is often required, such as ear notching. This method is likely to be painful and to date there is limited literature on pain assessment and alleviation for this routine husbandry practice. Here we aimed to determine if the mouse grimace scale (MGS) could be used to assess pain in C57BL/6 mice following routine ear notching. Langford et al. found that very acute noxious stimuli (i.e. < 10 min in duration) did not produce a change in MGS score in comparison to baseline. Here, no significant difference was found between MGS scores at baseline and immediately post ear notching, potentially indicating that the pain associated with ear notching is either too acute to assess using the MGS tool or the practice is not painful. Studies in other species indicate that ear notching is painful, therefore, unless we can confidently conclude that the process of ear notching is not painful, we should err on the side of caution and assume it is painful due to the large number of mice ear-notched and potential welfare consequences. Alternative methods of assessing pain following this routine practice should be used in order to assess both the potential pain in mice, and the effectiveness of analgesics or local anaesthetics to relieve any associated pain.