1 Results of enzyme-linked immunosorbent assays for bovine viral diarrhoea virus. http://ojvr.org/index.php/ojvr/article/downloadSuppFile/323/203
Of the 10 animals that tested positive during IHC staining, only six tested positive for viral antigen with the ELISA. PI animals will always be antigen positive, as they are consistently viraemic. This result indicated that animals 6, 7, 8 and 9 were not PI. These cases were re-evaluated, and as discussed later, they showed nonspecific positive staining in mast cells, leading to the false positive diagnosis. A PI animal will not develop antibodies against the strain of BVDV that infected it in utero (McClurkin et al. 1984). Of the six animals that tested positive for viral antigen, three did not have antibodies against BVDV, which confirms their PI status. However, if a PI animal is infected with a significantly different strain of BVDV after birth, it could develop antibodies to that strain (McClurkin et al. 1984). This explains why three animals that tested positive for viral antigen during ELISA and IHC staining, also tested positive for antibodies against BVDV. DiscussionIHC staining on ear notch biopsies is considered a fast and accurate diagnostic tool to identify animals that are persistently BVDV infected (Hilbe et al. 2007; Houe et al. 2006; Luzzago et al. 2006; Thur et al. 1996). The overall prevalence of PI calves entering feedlots in this study was 2.9%, which is much higher than the 0.5% estimate of Ishmael (2003). The estimate was based on the assumption that 1% PI animals are born into infected herds per year and that half of the animals are expected to die before weaning. Less than 0.5% of such animals will thus be available to enter feedlots owing to the early deaths of PI animals. Unpublished data from a South African study in July 2004 indicate a prevalence of PI animals of 0.56% in a single feedlot based on 2994 samples randomly collected from 20 000 calves entering the specific feedlot (W. Schultheiss, pers. comm., 01 September 2010). The samples in the present study were, however, taken from a selected population of calves, which were visually suspect animals and chronic poor doers, and therefore results cannot be directly compared to those of other studies. Feedlots receive large numbers of animals from a potentially large number of infected herds, increasing the possible number of PI animals that enter their feedlot population. Loneragan et al. (2005) state that when one PI animal is identified in a herd, the herd likely contains others as well and it is thus likely that PI animals entering a feedlot are clustered by herd of origin. In addition, many truckloads of calves entering South African feedlots are sourced from auctions and speculators, thus representing a collection of comingled animals from a variety of herds. Many of these high-risk calves are culled from breeding herds because of poor growth performance at weaning. A higher percentage of positive animals may thus be present in a feedlot compared to a single infected herd where, based on data from North America and Europe, approximately 1–2% of calves born are infected persistently. The samples were collected mainly by feedlot personnel and distinctions were not made between animals entering the feedlot for the first time and chronic poor doers. It is thus not possible to determine how many suspected PI first-time entrants and how many chronic poor doers were sampled. It was, however, clear from the samples received that feedlot personnel preferred sampling chronic poor doers compared to first-time entrants. Selection of first-time entrants was based on their physical appearance, including appearing pot-bellied, unthrifty and thin. The feedlots were specifically interested in the possible causes of chronic poor doers, as it is an important problem in the feedlot industry that results in financial losses. This suggests that the likelihood of a PI animal to become a chronic poor doer in a feedlot is increased, which may also have played a role in the higher prevalence (2.9%) observed in this study. Loneragan et al. (2005) found that PI animals were more likely to become chronically ill or die. In the group of animals entering the hospital pen for the first time (collected separately at the end of the study), only 1% were infected persistently according to IHC staining. The hypothesis that persistent infection with BVDV would increase the likelihood of contracting a respiratory illness soon after admission owing to the immunosuppressive nature of the disease was not substantiated during the study. However, the seasonal nature of bovine respiratory disease, the varying profile of calf ages and different purchasing strategies by the participating feedlots, coupled with the relatively small sample of hospital cattle in this study, preclude any firm conclusions on the contributory role of BVDV to bovine respiratory disease in South African feedlots. In addition, a significant number of calves that die following persistent infection with BVDV show severe complicated respiratory pathology with or without salmonellosis (D.J. Verwoerd, pers. comm., 03 February 2011). Further studies on the role of BVDV in the health of South Africa feedlot cattle are clearly indicated. Other factors causing respiratory disease in cattle in feedlots include the time of year (more prevalent in winter), extremely dusty conditions, stressors such as over-stocking, social structure in the pen, rain and mud, wind, poor adaptation, nonvaccinated (not pretreated) animals and respiratory disease following acidosis. In the unpublished study by Schultheiss mentioned earlier, no specific evidence of an increase in pneumonia in PI calves was found, but the presence of a PI animal in a pen increased the risk of the other calves in the pen contracting pneumonia threefold (W. Schultheiss, pers. comm.). This also corresponds to other studies such as that by Loneragan et al. (2005), who found an increase in respiratory disease in PI calves. The authors found the incidence of respiratory tract disease to be 43% greater in cattle with an opportunity for direct contact with a PI animal. These cattle also required more treatments for respiratory disease compared with cattle not exposed to a PI animal, thus incurring substantially higher medical costs.The reliability of IHC staining to diagnose PI animals was also tested in this study. One of the most important findings was that nonspecific staining may lead to diagnosing an animal incorrectly as being infected persistently with BVDV. This problem was investigated after an inappropriate number of positive cases were diagnosed. Nonspecific positive staining was observed in round as well as spindle-shaped mast cells. As the pathologist becomes more proficient in evaluating these sections, it becomes clear when mast cells are staining positive because the granules have a distinctly different shape from the granular virus staining. All sections diagnosed as positive were re-evaluated to exclude any false positives from the final results. Although nonspecific staining was observed with both stains, it was far more frequently seen with DAB staining. It is not certain why mast cell granules stain positively with the BVDV antibody. This phenomenon is not seen in every negative case and mast cells granules do not always take up stain. The possibility of over-staining should also be considered, as many of the affected sections showed colour changes in the connective tissue and cartilage, which is indicative of over-staining. All positively diagnosed animals must have positive staining in keratinocytes of the epidermis (stratum basale and stratum spinosum), hair follicle epithelium, smooth muscle cells of blood vessels and spindle-shaped cells in the connective tissue of the dermis (fibroblasts) (Njaa et al. 2000; also findings fromt his study). If these parameters are considered and cases are carefully examined, IHC staining can be considered to be a fast, reliable and cost-effective tool to identify PI animals. Cornish et al. (2005) found that the IHC stain detected 100% of PI calves. The monoclonal antibody used in the IHC staining method received from Cornell University was specifically developed for research purposes and is considered to be of the highest quality. All samples in this research project were stained with it. One of the limitations of this study was the inability to collect serum samples from all animals sampled. Serum samples for testing were available for only 10 PI animals as diagnosed according to IHC staining. Of these, six calves were positive for BVDV antigen (consistently viraemic) and three had antibodies to BVDV (Table 1). The IDEXX HerdCheck BVDV Antibody Test Kit has high specificity (> 99.7%) and sensitivity (nearly 100%) and detects BVDV types I and II antibodies (IDEXX Laboratories n.d.). However, it cannot be used on its own to identify a PI animal, for the following reasons. A PI animal will not have antibodies against the BVDV strain that infected it in utero because the immune system recognises the virus as self. Animals infected in utero after the immune system is competent can develop an antibody response, as can PI animals infected after birth with a different strain (McClurkin et al. 1984). They will, however, be antibody negative towards the strain that caused the initial infection. In this study three of the six PI animals showed the presence of antibody, indicating infection by a different strain of virus after birth. A PI animal will have a positive result with the BVDV antigen capture ELISA because it is persistently viraemic. An acutely infected animal will, however, also test positive, and this test is therefore not able to confirm PI status. The test is based on the robust Ems (gp48) antigen, which is consistently present in large quantities in both serum and tissue, making it easy to detect with the test kit. This antigen is highly stable, yielding reliable results even after long storage. Of the sampled animals, four were negative for antigen but positive for antibody, indicating that they were not infected persistently. Re-evaluation of the immunoperoxidase sections indicated that all four of these were incorrectly diagnosed originally as positive owing to the presence of nonspecific staining. Based on the information obtained in this study, IHC staining is a good and reliable tool to diagnose PI animals. The finding corroborates those of Cornish et al. (2005), Hilbe et al. (2007), Houe et al. (2006), Luzzago et al. (2006) and Thur et al. (1996). However, the pathologist evaluating these sections should be able to differentiate clearly positive from nonspecific staining according to the advised parameters. Too few skin samples were accompanied by serum samples to compare determinations by IHC staining and ELISA antibody and antigen statistically. In general, IHC staining is more cost effective, as only one test needs to be performed compared with both ELISAs. Currently the cost for one animal’s IHC stain test is approximately equal to the cost of one of the ELISAs. Conclusion The incidence of PI calves entering feedlots in South Africa is estimated to be approximately 0.56%. In this study the incidence was 2.9% because the samples were collected from a selected population of calves that appeared visually suspect or were recognised as poor doers. It was concluded that the possibility of a PI animal to become a chronic poor doer increases in a feedlot situation compared to calves raised extensively. This study confirmed the reliability of the IHC staining method to identify PI animals, despite the possibility of wrong diagnosis owing to nonspecific staining. Acknowledgements We would like to express our appreciation to the following people: Dr Sarah Clift for her support and help with interpretation of the IHC stains, and troubleshooting; Ms Vanessa Prinsloo for cutting of sections; Mrs Marie Smit for performing the IHC stains; Prof. J.A. 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