Monitoring and Investigating Natural Disease by Veterinary Pathologists in Diagnostic Laboratories

Laboratory-Based Monitoring of Emerging Disease

Some newly identified diseases that were recognized by veterinary pathologists will be familiar to readers of Veterinary Pathology: G. A. H. Wells at the Ministry of Agriculture, Fisheries and Food’s (MAFF’s) Central Veterinary Laboratory (CVL) in England and the emergence of bovine spongiform encephalopathy in the United Kingdom; T. McNamara with the Wildlife Conservation Society and the spread of West Nile virus to North America; E. S. Williams at Colorado State University and chronic wasting disease in captive and free-ranging deer; E. G. Clark and J. Ellis in the Western College of Veterinary Medicine in Saskatchewan and the recognition of postweaning multisystemic wasting syndrome in pigs. Interested readers might consult Saunders's biographical history of veterinary pathology to see how often creative investigation by diagnostic pathologists led to the characterization of new diseases. ²⁴ Table 1 lists factors that may facilitate this process. One example of effective pathology-driven diagnostic investigation is bovine neosporosis.

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Table 1.

Attributes of Veterinary Diagnostic Pathologists Who Identify New Disease Syndromes Through Routine Accessions

1. Respect for and ability to communicate with animal owners, wildlife managers, and clinical veterinarians

2. A collegial diagnostic team, including pathologists, in which curiosity, skepticism, open-mindedness, competence, and knowledge of the biomedical literature are valued

3. Access to institutional resources ($10,000 to $30,000) to offset initial testing costs

4. Access to adequate –80°C freezer space and long-term archives for wax blocks, glass slides, serum, and laboratory reports

5. Colocation in one building of diagnosticians, including epidemiologists, and basic researchers (particularly microbiologists)

6. Willingness to visit properties during unusual outbreaks of disease

7. Networking with fellow diagnosticians, clinicians, and basic researchers for ideas and advice

8. Solicitation of additional cases through responsible use of public and professional meetings, Web-based information, newsletters, press releases, and professional e-mail lists (eg, Listserv)

9. Familiarity with capabilities, limitations, and costs of other disciplines—particularly, classical microbiology, electron microscopy, epidemiology, and molecular diagnostics

10. Ability to identify and collaborate with basic researchers—ideally, ones who understand diagnostic and clinical medicine

11. Familiarity with material transfer agreements

12. Willingness to perform or facilitate transmission studies to meet Koch's postulates

13. Time and ability to report unusual disease episodes in peer-reviewed journals

14. Creative use of resources, such as serum banks, laboratory archives, diagnostic databases, hunter harvests, and slaughterhouses

15. Administrative recognition of the value of using spontaneous disease as springboard for building client support, contributing to lay and peer-reviewed publications, and developing diagnostic experience

Case Example: The Recognition of Neospora caninum Abortion in Cattle

Neosporosis is now recognized as an important cause of abortion in dairy cattle worldwide. The causative agent, Neospora caninum, was identified but not named when pathologists at the Norwegian College of Veterinary Medicine reported encysted sporozoa in a canine encephalomyelitis and myositis syndrome. ^8,9 Earlier reports recorded a similar agent in calves in New Zealand, Australia, Italy, the United States, and England. ²¹ The disease in cattle was variously identified as toxoplasmosis, toxoplasmosis-like, sarcocystosis, and sarcocystosis-like. It is likely that many diagnostic pathologists saw the lesions of neosporosis before the 1980s. They either did not recognize their significance or elected not to pursue the problem. For example, MAFF diagnosticians at the CVL in Weybridge, England, and in regional disease investigation centers in England and Wales recognized protozoa in the brains of aborted and congenitally abnormal calves throughout the 1980s. Characterization was hampered by two factors. First, MAFF’s disease investigation system had a spoke-and-wheel organization. Almost all postmortem examinations were performed in veterinary investigation centers. Depending on the capabilities of the center, fixed tissues were either examined in-house by veterinarians with some training in histopathology ¹⁶ or submitted to pathologists at the CVL who contended with the disadvantages of proxy necropsies. There was little incentive for MAFF pathologists to visit source properties or talk to practicing veterinarians. Second, the organization of MAFF diagnosticians, along disciplinary lines in separate buildings at the CVL, limited interaction among diagnostic pathologists, microbiologists, and epidemiologists. As a result, no interdisciplinary team was established to pursue a recurring disease syndrome unless MAFF was confronted with something truly awful, such as bovine spongiform encephalopathy. It is one thing to see a distinctive lesion or disease once or twice; but when seen recurrently, it cries out for something more than a report stating, “We see this all the time and are uncertain of its significance.”

The same lesions were seen by diagnostic pathologists at the California Veterinary Diagnostic Laboratory System (CVDLS; now the California Animal Health and Food Safety Laboratory System) shortly after a branch diagnostic laboratory was opened in Tulare, California. Tulare’s pathologists quickly identified it as a distinctive abortion syndrome in drylot dairies. At the time, veterinarians in San Joaquin, California, were so familiar with abortion rates approaching 20% that they began to wonder whether they might be normal. Diagnostic pathologists M. L. Anderson, B. C. Barr, and P. C. Blanchard performed necropsies on fetuses and routinely sampled brains as well as skeletal muscles. This was at a time when few laboratories did this, because it was considered futile to examine autolysed brains. The pathologists recognized that many aborted calves had distinctive nonsuppurative lesions, many of which contained intralesional protozoa. ^1,2 Blanchard noticed a resemblance to the lesions of toxoplasmosis but ruled out that disease by serological testing. Barr undertook a retrospective study of archived slides and established that protozoan cysts were present in a high proportion of affected fetuses. The suspicion that a new agent was involved was strengthened when J. P. Thilsted in a New Mexico diagnostic laboratory reported a similar syndrome. ²⁶ Some years earlier Thilsted’s coauthor J. P. Dubey isolated and named N caninum after examining dogs with a neurological disease. ^12,13 The report by Thilsted and Dubey was the first to establish a role for N caninum in bovine abortion.

The New Mexico investigation, supplemented by reports from Washington and Maryland, ^14,15,23,25 encouraged Anderson, Barr, and Blanchard to report neosporosis as an important cause of bovine abortion in California. ¹ The CVDLS pathologists involved Dubey and another veterinary parasitologist, D. S. Lindsay, who provided advice and reagents. They were fortunate that Lindsay had recently described the in vitro development of N caninum and published a method to demonstrate the agent in fixed tissue. ^17,18 The salient diagnostic lesions and the ultrastructural appearance of the organism in bovine fetuses were well illustrated in a series of publications from the CVDLS, alerting diagnosticians elsewhere to the syndrome. ^1-3,5,6 The CVDLS’s director, A. Ardans, recognized that a successful investigation of the syndrome would demonstrate the value of the laboratory network to clients and policy makers. Ardans waived testing costs and encouraged diagnosticians to present their findings to veterinarians at county meetings. These meetings were helpful for soliciting cases of additional outbreaks and for exchanging information with clinicians who worked with the disease firsthand. The CVDLS provided laboratory space for a veterinary protozologist, P. A. Conrad, so that isolation of the protozoan could be attempted. A university epidemiologist, M. Thurmond, and a CVDLS molecular diagnostician, S. Hietala, began working with the pathologists. After processing more than 100 brains supplied by diagnostic pathologists from throughout the CVDLS system, Conrad’s laboratory isolated N caninum. ¹⁰ Isolation of the protozoan facilitated the production of improved diagnostic reagents, as well as experimental reproduction of the disease. Although hesitant at first to declare that the “Neospora-like” agent from cattle was identical to N caninum in dogs, ^5,6 it gradually became clear that they were the same. Koch's postulates for its role in bovine abortion were met in 1994. ⁷  A useful immunofluorescent antibody test developed by Conrad was supplemented by an enzyme-linked immunsorbent assay developed in Hietala’s laboratory in 1995. ²² Serological tests facilitated larger-scale surveillance of infected herds. The CVDLS used this approach to assess the relative roles of vertical and horizontal transmission and the conclusion that vertical transmission was important. ⁴  A pathologist working in the California system and later in Wyoming, M. M. McAllister, was stuck by the apparent point source nature of some outbreaks. This prompted him to wonder whether dogs served as definitive host species. McAllister confirmed his suspicion in 1998. ¹⁹

The recognition of neosporosis in bovine abortion was triggered by routine laboratory submissions, but the process that it catalyzed was not passive. Once they recognized that something unusual was occurring, CVDLS pathologists solicited cases, visited dairies, exchanged ideas with veterinarians and dairy farmers, preformed retrospective studies of archived cases, compared the ultrastructure of the Neospora-like agent from cattle to that of N caninum from dogs, encouraged the development of laboratory tests, published findings, and worked with anyone who might help, including epidemiologists, molecular diagnosticians, serologists, parasitologists, and graduate students. Many diagnostic laboratories are efficient at generating reports but tend to defer to owners or veterinarians when they see something puzzling. Others, such as the CVDLS, accept the challenge of actively pursuing emerging disease syndromes and encourage laboratory personnel to take the initiative. Of course, laboratories must strike a balance between routine diagnosis and an investigative mode. But any laboratory that does not initiate disease investigations from time to time will lose touch with its client base. The bond between laboratories and clients is already tenuous owing to the anonymity of faxed/Web-based pathology reporting and the ease with which diagnostic samples can be shipped nationally. Diagnostic pathologists and their diagnostic colleagues tend to develop strong rapport when dispatched to a farm or ranch to conduct an on-site investigation. It is effective public relations for the laboratory; it is good mental preparation for a serious disease outbreak; and it is stimulating, educational, and—dare one say it—fun. There is a reason the Journal of Veterinary Diagnostic Investigation was so called and appropriate because much of the early work on bovine neosporosis was published in that journal. It was a model of integrated disease investigation by diagnosticians.

Sustaining Disease Investigation by Diagnostic Pathology Services

Some diagnostic veterinary pathologists assume that the heyday of the discipline has passed and that hard-won skills are being made redundant by molecular diagnostic medicine. Sometimes this comes with a fear-biting attitude to molecular diagnosticians. Yet new diseases continue to be presented to pathology services, and diagnostic pathologists occupy the strategic nexus to pursue them, provided they take the initiative. The increased importance of molecular diagnostics means that diagnostic pathologists need to understand basic principles of diagnostic test validation and accuracy, as well as the strengths and weakness of individual assays. For their part, molecular biologists who are willing to work on natural diseases can use diagnostic pathologists as a bridge to spontaneous outbreaks. Vibrant long-term investigative/experimental partnerships can be forged between molecular biologists and diagnostic pathologists, as occurred in bovine neosporosis. It helps when diagnostic pathologists understand what academic collaborators in the basic sciences need when they accept the challenge of working on an emerging disease: extramural grants, high-impact publications, ideas for graduate students, veterinary input, and proof from the field that the problem matters.

I teach an undergraduate course in diseases of food animals and horses. Students are shown, at the start of each semester, a list of “new” diseases identified since I graduated as a veterinarian in 1977. This is done to alert the class that, whereas common things are common, each year a typical state- and province-funded diagnostic laboratory sees at least one new disease. Many are minor, such as a novel genetic disease or a familiar infectious agent in the “wrong” species. But most diagnosticians should expect to encounter, at least once in their careers, an emerging disease of regional, national, or international significance. It should be part of their training to be alert to the encounter and to know what to do when it happens. Diagnostic veterinary medicine in North America has two strengths that are missing in much of the world: First, its laboratories employ trained pathologists, rather than veterinary generalists; ¹⁶ second, there is such diversity of institutional cultures between laboratories. It is noteworthy that in spite of a large private-laboratory sector, so many new diseases continue to be identified in diagnostic submissions by workers in state-funded diagnostic laboratories—particularly, university-affiliated ones. This exercise requires discretionary investment, such as the perennially threatened 1887 Hatch Act funds. These earmarked federal funds support applied agricultural research in land grant colleges in the United States and are often tapped to defray part of the cost of testing early in a disease investigation. An institutional culture that values field investigation among its personnel is critical, particularly when employees are assessed for promotion. It is helpful when diagnosticians and basic scientists share a common workspace so that ideas and observations can be shared and challenged.

Veterinary pathologists can make good field investigators, particularly when they have a clinical background and an awareness that they hunt best in collegial packs. It helps when they are in a workplace that encourages creative use of diagnostic accessions. Perhaps the American College of Veterinary Pathologists and the American Association of Veterinary Laboratory Diagnosticians should consider jointly offering a short course in disease investigation by diagnostic teams, focused on the role of pathologists. Regardless of formal training, there is no substitute for encouraging veterinary pathologists, especially when newly minted, to think bigger than the role of simply turning cases around quickly and efficiently. Sometimes it is the lesion that causes a pathologist to pause and curse his or her luck at seeing something unfamiliar that opens the door to a new disease.