Abstract

Sera from three species of dolphin were examined over a period of two to three years by a reference complement fixation test for antibody responses to a range of viral antigens and to Mycoplasma pneumoniae. Despite the presence of non-removable anticomplementary factors in all dolphin sera, an evaluation method was devised to yield valid antibody response curves. Serum antibody responses to M. pneumoniae and Respiratory Syncytial Virus could be correlated with disease episodes and also with current activity of those agents in the local population.

Introduction

Respiratory diseases are common amongst the three species of dolphin (Tursiops aduncus, Tursiops truncatus, and Lagenorhynchus obscurus) held in captivity at the Durban Dolphinarium. Episodes of respiratory illness would often spread rapidly amongst the dolphins (frequently with the trainers complaining of a concomitant pulmonary involvement). The episodes appeared to be seasonal, tending to occur more freqtipntly in the Winter nionths. Bacteria cultured from blowhole "cough" plates varied from dolphin to dolphin and from season to season. It was thus postulated that a virus may be incriminated as the cause of the initial primary insult to the respiratory mucosa. In order to establish this, apart from haematology and biochemistry, the serum was analyzed by means of the complement fixation test to detect increases in the serum viral antibody titres.

Materials and Methods

Specimen Collection, Preparation, and Storage

Specimens consisting of 5-10 ml of clotted venous blood, collected aseptically from the central venous plexes in the tail fluke, were spun at 3000 rpm for 10 min in a Sorval GLC-4 centrifuge and the supernatant serum was transferred to 2 ml glass vials with rubber-lined caps and stored at -10ºC.

Complement -Fixation rest (CFT)

The sera were tested using the Center for Disease Control Laboratory Branch Complement Fixation Reference Test (1). This standardized method ensures reproducibility of results and permits comparison between results obtained over a prolonged period of time and with different batches of test reagents.

Due to the non-availability of specific viral antigens for viruses of dolphins, the sera were tested in parallel with human sera by routine hospital viral diagnostic screening for endpoint titres of antibodies reacting with human viral antigens of Influenza A, Influenza R, Parainfluenza (1, 2, and 3), Respiratory Syncytial Virus (RSV), Adenovirus, Measles, Mumps, Varicella-Zoster, Herpes Simplex, Cytomegalovirus, and also Mycoplasma pneumoniae.

Anticomplementary Factors in Dolphin Sera

It was unfortunately found that the dolphin sera regularly exhibited fairly high levels of anticomplementary (AC) activity.

The degree of AC activity varied from dolphin to dolphin and also from time to time in the same dolphin. In general, whenever the testing schedule necessitated refreezing and rethawing of a serum specimen, its AC titre rose by a factor of 2-4. Attempts were made to eliminate the AC activity by preincubation of the serum with undiluted complement, by the use of additional electrolytes in the form of magnesium chloride and calcium chloride, by the addition of potassium citrate to effect deaggregation, and by prior treatment of the serum with an ion-exchange resin.

However, no single treatment was regularly effective in eliminating AC activity in all dolphin sera, and it was concluded that the factors involved might he a characteristic of the species. As the test method used was evolved specifically for testing human sera, modifications of the basic method using different combinations of complement, erythrocytes, and haemolysin from various animal species might he required in order to obtain an optimal CFT system for the routine testing of dolphin sera.

Despite the presence of this non-removable AC factor in the dolphin sera, it was decided, nevertheless, to continue using the viral CFT monitoring system and to simply record both the AC value and the specific test value of the sera for each test run. This procedure was adopted in the hope that any specific rise in antibody level would be clearly distinguishable from the background AC level seen for each dolphin, and has subsequently been continued over a period of 2-3 years for each dolphin.

Interpretation of Results

In order to interpret the results, three sets of graphs were drawn. These are discussed in detail by Thurman & Windsor (in press). On evaulation of the graphs, it appeared that three of the dolphins experienced one or more episodes of Mycoplasma pneumoniae infection. One particular dolphin also underwent an episode of RSV immediately following the M. pneumoniae infection. There also appears to have been some measles virus activity immediately prior to the M. pneumoniae episode, although in this case the peak titre did not rise as high as that exhibited following infection by M. pneumoniae, and a possible boost by re-exposure to measles may have occurred a few months later.

Discussion

The CFT is laborious and time-consuming, and when AC factors are present, results are at best difficult, and in many instances, impossible to interpret. Such factors are found in approximately 4% of human sera submitted for hospitalized patients (3) and were found in all members of the three species of dolphin studied.

In the case of patient sera, usually one or two specimens are submitted for each patient within a short period, and if AC factors are present which are not removable by preincubation with excess complement, no further attempts are made to utilize the specimen in this test.

However, the CFT still offers many advantages in virus serology over newer methods such as radioimmunoassay, enzymeimmunoassay, or fluorescent antibody testing. For instance, it is still possible to test a wider range of virus antigens by CFT than by any other single method, using commercially available reagents.

There are many variations of CFT methodology, and the main advantage of the Reference CFT ties in the high level of reproducibility of results. Repeat tests generally yield the original result or vary by only a single 2-fold dilution endpoint from the original. In addition, due to the precise standardization of the indicator system and exact antigen calibration, it is possible to continue a survey over a number of years using reagents from various sources and yet retain complete comparability of results.

For these reasons, it was decided to use the Reference CFT for the testing of dolphin sera despite the difficulties encountered with the AC factors and, when results were evaluated over a prolonged period of time, it was found possible to derive antibody profiles, as explained above, for each dolphin for each of the viruses studied.

The viral antigens used were those of human viruses, although it was originally unknown whether or not the dolphins would be susceptible to viruses of man, whether any such infections would lead to disease episodes, and what type of antibody response Might lead from exposure to these viruses. Alternatively, it was hoped that if dolphin-specific viral infections occurred, there might be sufficient cross-reactivity between related dolphin and human viruses to produce a recognizable reaction in the tests.

The response to M. pneumoniae antigen is quite definite for all three dolphins, reaching levels far in excess of the median titre of 32 established for hospitalized patients in Durban during the same time period, and approximating levels reached in patients with confirmed A. pneumoniae infection (4).

As the dolphins all underwent a respiratory disease episode conforming to the antibody response curve and responded to specific antimycoplasma therapy, it is considered that these interpretations do, in fact, present an accurate picture of their immune, response to infection.

Moreover, a three-year survey of respiratory infections in hospitalized patients (4) showed that M. pneumoniae was the most important non-bacterial respiratory disease agent in the Durban area for the years 1980-1982, with peak activity in 1982. It therefore appears likely that the antibody responses of the dolphins were to infection with the human agent and not to cross-reactions occurring with dolphin-specific strains of mycoplasma.

One dolphin, "Bilbo", since deceased, apparently experienced a series of viral infections during the course of the study. His results show an undoubted response to RSV infection following M. pneumoniae; the peak titre of 1024 being far above the median of 16 seen in hospitalized patients, and corresponding to the highest values recorded for children with severe RSV infection. Again, RSV is an important respiratory disease agent in the Durban area, being only slightly less important than Adenovirus in young children.

Elevated serum titres obtained for Measles virus in this dolphin did not correspond with a well-defined clinical episode and may have represented response to asymptomatic infection or re-exposure. It is unquestioned that exposure to Measles virus is probable in the Durban area where measles still ranks high on the list of notifiable diseases, and is particularly important in young children in predisposing to infection with other respiratory viruses.

In conclusion, it can be said that the method of testing and the interpretation of results reported here represent a valid means of monitoring respiratory infections in dolphins. Moreover, it appears probable that human respiratory agents which are prevalent in the community may represent a risk to dolphins which have had no previous experience of these agents.

References

1. U.S. Public Health Service. Standardized diagnostic complement fixation method and adaptation In micotest (Public Health Service publication - 1228). Washington D.C., U.S.A., Government Printing Office (1965).
2. Windsor, I.M. and Thurman, G.D. Elevation in serum antibody titres in captive dolphins following respiratory disorders caused by viruses and Mycoplasma pneumoniae. Investigational Report, Oceanographic Research Institute.
3. Lennette, E.H. and Schmidt, N.J. In: Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections. American Public Health Association (1979).
4. Windsor, I.M. A three-year survey of respiratory virus infections in Natal. Paper presented at the 3rd Annual Congress of the South African Society for Microbiology, April, 1984 (1984).