Using the PCR primers designed here, we were able to develop a sensitive and specific test for P. neurophilia
, which yielded a 788-bp fragment with no extraneous reaction products. The PCR primers Pn18S5F and Pn18S5R were designed to be specific to P. neurophilia
based on DNA sequence alignment of SSU rDNA from 48 other microsporidia, including Loma salmonae
, a representative of a closely related genus.18
Indeed, specificity of the PCR test was high, and it did not cross-react with any other piscine microsporidia available to us (). Although the PCR test did not detect these other microsporidians, we were unable to check the specificity of the PCR primers against all known parasites or against as-yet unknown parasites that might emerge and that could share similar DNA sequence with P. neurophilia
. Therefore, it is advisable to periodically confirm the infection status in a proportion of fish by using an alternate technique, such as DNA sequencing. This confirmation may be especially important in facilities where the parasite has not been detected previously.
Specificity of ribosomal DNA polymerase chain reaction with microsporidian general primers 530f/580r and Pseudoloma neurophilia-specific primers Pn18S5F/Pn18S5R for detection of microsporidia
The method used for sample preparation from tissues is important for recovering DNA from resilient microsporidian spores. Indeed, extraction of DNA from intact spores may require physical disruption, as suggested by Müller and coworkers.20
However, Docker and colleagues8
obtained equivalent results in detection of Loma salmonae
when tissues were processed by disruption with silica beads, mechanical homogenization, or proteinase K digestion alone. Because Docker and coworkers8
found Proteinase K digestion to be the least labor-intensive and less subject to cross-contamination than the other methods tested, we too used Proteinase K digestion that was preceded by an overnight freeze–thaw. This method also was preferred because it required equipment that is available in many laboratories. In addition to using a straightforward method of DNA extraction, we also avoided the use of a nested PCR test. Although a nested PCR test may increase sensitivity,3
the likelihood of false positives also increases.4
The lower limit of detection of this test with DNA originating from infected spinal cords or purified spores was consistently 10 spores or fewer per PCR reaction (). Detection of a
single spore in a PCR reaction was inconsistent; we obtained positive results from 1 of 4 (25%) reactions originating from spores, and positive results from 2/4 (50%) reactions originating from infected tissue. Occasionally, results were positive for infected tissues diluted to a theoretical 0.1 spores per PCR (2 of 4, 50%; ).
Detection of a fraction of a spore is possible, as multiple copies of the target gene occur in each spore. Differences in detection level between purified spores and infected tissues can be explained by a number of factors. For example, our estimate of the number of spores present in the infected tissue ‘slurry’ was based on the number of spores we purified per tissue volume. The loss of spores during the purification process likely would make our estimates of the number of spores in the tissue low, as tissues may contain developmental stages that were not counted. This difference would increase the apparent sensitivity of the PCR test on tissues as opposed to suspensions of pure spores.
Sensitivity of the polymerase chain reaction with Pn18S5F/Pn18S5R primers from infected tissues and purified spores (number positive/number tested)
Polymerase chain reaction tests have inherently low limits of detection, and the detection of 10 spores (sometimes 1 or 0.1) we obtained for P. neurophilia is consistent with other studies of fish microsporidia. Using spore number estimates from infected tissues, Docker and colleagues8
were able to detect as few as 0.01 to 0.001 Loma salmonae
spores from gill tissue and 0.1 Nucleospora salmonis
spores from infected kidney tissue.9
Barlough and coworkers3
reported that 10 infected lymphocytes were needed for a positive reaction in their PCR test for N. salmonis
, which may equate to as few as 10 parasites.
Although these estimates usually are based on detection of spores, it is essential to note that PCR can detect stages otherwise undetectable by histology. For example, PCR will detect developmental stages of a parasite early in the infection process, when abundance is low.16,19
Furthermore, as entire fish or whole spinal cords are used for DNA extraction, our sampling regime is unlikely to overlook P. neurophilia
infections due to unequal distribution of the parasite, especially during these earlier stages of infection. It is difficult to directly compare our findings from PCR with histology, as whole fish are used for histology, leaving no tissue for PCR. Likewise, removal of the spinal column and brain or use of whole fish for PCR leaves little tissue for histology. Ultimately, the choice of diagnostic technique depends on the goals of each individual study. Histological analysis is more appropriate for evaluating pathologic changes, whereas PCR is better for screening large numbers of samples, subclinical populations, small young fish, water, and other materials.
Presence of P. neurophilia
within water systems or associated with biofilms has yet to be demonstrated. However, water collection and DNA extraction methods that have been developed for detection of medically important water-borne parasitic protozoa such as Cryptosporidium parvum
, Giardia intestinalis
, and other microsporidians12,21
can be used for P. neurophilia
. The observation by Kent and Bishop-Stewart17
of P. neurophilia
spores in ovaries and occasionally eggs of zebrafish raises concerns about spread of this parasite through transplantation of infected eggs. Hogg and colleagues15
documented vertical transmission of microsporidia in amphipods by using a PCR test, and we currently are investigating this phenomenon with P. neurophilia
in zebrafish. Until vertical transmission can be verified, screening of eggs by PCR may be unwarranted at this time, although standard quarantine procedures should still be implemented for fish and eggs introduced to research facilities.
The described PCR test may prove useful for screening fish prior to introduction to new facilities. Although pathogen screening protocols are applied routinely to fishes used in aquaculture and rodents used in research, this approach has yet to be adopted by the zebrafish research community. Furthermore, no chemotherapeutant against P. neurophilia
has yet been developed; therefore monitoring and preventing movement of infected fishes between facilities is preferred to control the spread of the parasite at this time. The number of fish to be tested from an incoming shipment depends on the predicted prevalence within a normal population.2
In our evaluation of the P. neurophilia
PCR test on 30 zebrafish from a population exhibiting no signs of clinical disease, we noted a 10% prevalence of the parasite. Therefore, with a predicted prevalence of 10%, between 20 and 30 fish (depending on the size of the population) would need to be tested to have 95% confidence that the parasite is absent from the population.2
We provide here a sensitive and specific test for P. neurophilia to be used by the zebrafish research community to monitor existing stocks of fish and avoid introduction of this common parasite. The use of PCR in research has become commonplace in most biological research facilities, and equipment is usually available. Therefore, the P. neurophilia PCR assay can be performed within individual laboratories by the researchers themselves to minimize costs. With the increasing concern for spread of this prevalent pathogen and the use of pathogen-free stocks of fish for experiments, this PCR assay is an excellent tool for diagnosis and can be adapted to any research situation.