3.4. Evidence of S. neurona in fecal samples from sea lions

3.4.1. Cell culture and serological examination

In an effort to propagate and isolate coccidian protozoa identified by light microscopy in sea lion feces, sporulated oocysts from CSL10089 (Coccidia A), CSL10092 (Coccidia A & B), CSL10100 (Coccidia A & B), and CSL10266 (Coccidia A) were prepared and inoculated into flasks containing MA104 cells. All cultures were fed and examined for the presence of live coccidian zoites three times per week. After 14 days, 21 days and 35 days of observation, cell cultures inoculated with sporulated oocysts from CSL10089, CSL10100, and CSL10092, respectively, showed propagating organisms with morphologic similarity to Sarcocystis spp. (Table 4). Parasites were observed both extracellularly and within MA104 cells. Extracellular zoites were 8 ‾ 11 M m in length and 1 ‾ 2 M m in width when measured with SPOT™ Advanced software at 200× magnification. Schizonts were seen periodically as the culture matured and the number of free zoites increased; suggesting that these were merozoites produced by schizogony. Free zoites had active, circumaxial motility characteristic of S. neurona merozoites observed in vitro (Miller et al., 2001a). Similar organisms did not grow in cultures inoculated with sporulated oocysts from a fourth sea lion, CSL10266 or in un-inoculated cultures of MA104 cells (Table 4).

a Oocysts were identified by microscopy during isolation, but DNA was insufficient for molecular characterization.

b The amplified ITS-1 region of ̃Ano11 is genetically distinct from other coccidian isolates (See Fig. 1).

c The presence of Sarcocystis neurona -like DNA was confirmed by PCR and sequence analysis. Sporocysts were not identified in fecal samples by microscopy.

By IFAT, antigen slides prepared from zoite cultures of all three sea lions, CSL10089, CSL10092 and CSL10100, tested positive using serum from a horse with equine protozoal myeloencephalitis (Fig. 5A ‾ C, Table 4). The horse's S. neurona infection was confirmed by IFAT and western blot on serum (1:320 titer to laboratory strain snUCD-1) and cerebral spinal fluid, and by immunohistochemistry used to detect S. neurona merozoites in the lumbar spine. In addition, all three zoite isolates from sea lion oocyst cultures reacted to sera from hospitalized sea lion CSL9878 (Table 4) which was itself seroreactive to merozoites of snUCD-1 (1:2560) (Table 1 and Fig. 5D). Sea lion culture antigen was IFAT-negative (<1:40) to goat serum containing T. gondii -reactive antibodies, bovine fetal serum containing N. caninum -reactive antibodies, and, as shown in Fig. 5E, to horse serum that was seronegative to S. neurona and N. hughesi. 3.4.2. PCR and sequence analysis

As soon as zoites were observed, supernatant from the MA104 cell cultures inoculated with sporulated, excysted oocysts from the fecal samples of CSL10089, CSL10100 and CSL10092 were collected for molecular characterization approximately once per week for 11 ‾ 14 weeks. Pan-coccidian primers that span the ITS-1 region produced a ~1000 bp band when DNA was amplified from zoites taken from cultures from the 3 sea lions throughout their collection period. This is the expected band size for amplification of S. neurona DNA using pan-coccidian ITS-1 primers (Gibson et al., 2011). In addition, a second band ~400 bp in size was amplified simultaneously in culture supernatant of CSL10100 and CSL10092 using the pan-coccidian primers, but only in the first week of sample collection following zoite observation (Table 4). Four hundred basepairs is the expected band size for amplification of Coccidia A, B or C using pan-coccidian ITS-1 primers (Colegrove et al., 2011; Gibson et al., 2011). In culture CSL10266, in which no zoites (only oocysts) were observed, only the ~400 bp band was amplified by PCR during the first two weeks of incubation. Subsequent supernatant samples of culture CSL10266 and all uninoculated MA104 control cultures were PCR-negative (Table 4).

Sequence analysis confirmed that the ~400 bp amplicon DNA generated in early sampling of cultures CSL10100, CSL10092 and CSL10266 was identical to DNA amplified from frozen, pelleted oocysts originally harvested from the same animal; namely, Coccidia A alone (in the case of CSL10092 and CSL10266) or a dual infection with Coccidia A and B (in the case of CSL10100) (Table 4). By BLAST analysis, good quality partial sequences (~700 bp; abbreviated due to the presence of a homopolymer region) generated from the ~1000 bp amplicon in CSL10089, CSL10092 and CSL10100 zoite cultures were 99% similar to S. neurona DNA isolated from horses (AF204230 & AF081944) and from a skunk (AY082648) (See Supplementary data). Dinucleotide mixtures in the ITS-1 sequences indicated the presence of at least two S. neurona -like genotypes in the sea lion cultures. These polymorphic regions shared nucleotide similarity to horse strains snUCD-1 (AF081944) and SN-MU1 (AF204230), sea otter strains SO3639 (DQ084486) and SO5259, and a skunk isolate (AY082648) (See Supplementary data).

NT = not tested.

a Coccidian species determined based on analysis of DNA from frozen pelleted oocsyts from feces and not culture inoculum, using ITS-1 locus PCR and sequencing.

b Based on genetic similarity across unambiguous sequence.

c 400 bp amplicon was only observed the first 1 ‾ 2 wks post-inoculation. The 1000 bp PCR products were amplified continuously in CSL10089, CSL10092, and CSL10100 cultures.

To further characterize the S. neurona -like organisms cultured from sea lion feces, we amplified seven additional genetic loci in DNA extracted from zoites in the sea lion oocyst culture supernatant. Table 4 displays the PCR and sequencing results for these additional loci. Using ITS 1500 primers, with known specificity for S. neurona or Sarcocystis falcatula (Gibson et al., 2011), a 500 bp product was consistently amplified in all culture supernatant samples from CSL10089, CSL10092, CSL 10100 in which zoites had been observed. By BLAST analysis, good quality partial ITS 1500 amplicon sequences (~300 bp, abbreviated due to the presence of a homopolymer region) were 98% identical to S. neurona isolated in Southern sea otters including isolates SO3528, SO3539, SO3485, and SO3501 (GenBank DQ 084485 ‾ DQ084488). Microsatellite primers amplified ~150 bp products containing di-nucleotide repeats CA 17 (Sn7) and GT 18 (Sn9) found in S. neurona strain SN-MU1 and other S. neurona strains isolated from opossums, sea otters, and cats (Rejmanek et al., 2010). Merozoite surface antigen SAG1 gene sequences of CSL10089, CSL10092 and CSL10100, amplified by SnSAG 1-5-6 primers, were 100% identical across 959 bp to S. neurona strain snUCD-1 (AF401682) (Ellison et al., 2002) and 99% similar across 968 bp to the representative sequence of the snSAG1 allele amplified in samples from terrestrial and marine mammals (GQ851951) (Wendte et al., 2010). Sea lion cultures CSL10089, CSL10092 and CSL10100 were identical to each other across a 948 bp sequence of the merozoite surface antigen gene SnSAG4 and 100% identical to sequences of S. neurona isolated from a sea otter (SO3106, GQ851957) and an opossum (OP134, GQ386979).

A unique sequence in sea lion S. neurona -like isolates was identified in the merozoite surface antigen 3 (SnSAG3) locus in which CSL10089 was confirmed by bi-directional sequencing to contain an AT insertion at positions 519 ‾ 520 (Table 5). The same insert was observed in single direction sequencing for CSL10092 but could not be confirmed in CSL10100 due to poor sequence quality. The same insert was not identified in SnSAG3 sequences available in GenBank originating in a variety of host species including sea otters, opossums and horses (Table 5). Lack of the position 519 ‾ 520 insert in laboratory strain SnUCD-1, derived from a horse in California (Table 5), ruled out the possibility that S. neurona growth in sea lion oocyst cultures was due to laboratory contamination. At other polymorphic SnSAG3 nucleotide positions, including 239 and 1059, sea lion S. neurona -like isolates were similar to published opossum and sea otter isolates (Table 5).