Toxoplasma gondii seroprevalence and association with equine protozoal myeloencephalitis: A case-control study of Californian horses.

While toxoplasmosis is not commonly considered a clinical disease of equines, previous seroprevalence studies have reported differing background rates of Toxoplasma gondii infection in horses globally. The objective of this study was to evaluate possible associations between T. gondii seroprevalence and clinical signs of equine protozoal myeloencephalitis (EPM) in horses. Using a case-control study design, 720 Californian horses with neurologic signs compatible with EPM were compared to healthy, non-neurologic horses for the presence of T. gondii antibodies (using indirect fluorescent antibody tests [IFAT]). Toxoplasma gondii seroprevalence among cases and controls was determined at standard serum cut-offs: 40, 80, 160, 320, and 640. At a T. gondii titre cut-off of 320, horses with clinical signs compatible with EPM had 3.55 times the odds of a seropositive test compared to those without clinical signs (P<0.01) when adjusted for covariates. When restricted to the autumn season and at the same titre cut-off, an EPM suspect horse had 6.4 times the odds of testing seropositive to T. gondii, compared to non-neurologic horses. The association between high T. gondii titres and clinical signs compatible with EPM is potentially reflective of toxoplasmosis in equines. Serologic testing of cerebrospinal fluid and isolation of T. gondii in EPM suspect cases should be considered. Future studies investigating the relationship between T. gondii and EPM are warranted.

Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts.

Two major obstacles to conducting studies with Toxoplasma gondii oocysts are the difficulty in reliably producing large numbers of this life stage and safety concerns because the oocyst is the most environmentally resistant stage of this zoonotic organism. Oocyst production requires oral infection of the definitive feline host with adequate numbers of T. gondii organisms to obtain unsporulated oocysts that are shed in the feces for 3-10 days after infection. Since the most successful and common mode of experimental infection of kittens with T. gondii is by ingestion of bradyzoite tissue cysts, the first step in successful oocyst production is to ensure a high bradyzoite tissue cyst burden in the brains of mice that can be used for the oral inoculum. We compared two methods for producing bradyzoite brain cysts in mice, by infecting them either orally or subcutaneously with oocysts. In both cases, oocysts derived from a low passage T. gondii Type II strain (M4) were used to infect eight-ten week-old Swiss Webster mice. First the number of bradyzoite cysts that were purified from infected mouse brains was compared. Then to evaluate the effect of the route of oocyst inoculation on tissue cyst distribution in mice, a second group of mice was infected with oocysts by one of each route and tissues were examined by histology. In separate experiments, brains from infected mice were used to infect kittens for oocyst production. Greater than 1.3 billion oocysts were isolated from the feces of two infected kittens in the first production and greater than 1.8 billion oocysts from three kittens in the second production. Our results demonstrate that oral delivery of oocysts to mice results in both higher cyst loads in the brain and greater cyst burdens in other tissues examined as compared to those of mice that received the same number of oocysts subcutaneously. The ultimate goal in producing large numbers of oocysts in kittens is to generate adequate amounts of starting material for oocyst studies. Given the potential risks of working with live oocysts in the laboratory, we also tested a method of oocyst inactivation by freeze-thaw treatment. This procedure proved to completely inactivate oocysts without evidence of significant alteration of the oocyst molecular integrity.

Endogenous transplacental transmission of Neospora hughesi in naturally infected horses.

Over a 2-yr study period, we investigated possible endogenous transplacental transmission of Neospora hughesi in 74 mare and foal pairs following the diagnosis of neuronal neosporosis in a weanling foal. Presuckle and postsuckle serum of each foal, serum and colostrum of each periparturient mare, and serum of each mare and foal pair, collected at 3-mo intervals thereafter, were tested for N. hughesi using an indirect fluorescent antibody test (IFAT). Furthermore, whole blood and colostrum samples and placentae were tested for the presence of N. hughesi by real-time PCR. The mares' seroprevalence at foaling based on IFAT (titer ≥ 160) was 52 and 6% in 2006 and 2007, respectively. Colostral antibodies against N. hughesi were detected in 96 and 11% of the mares in the 2-yr study. With the exception of 3 foals, all remaining foals were born seronegative to N. hughesi. Passive transfer of colostral antibodies to N. hughesi was documented in 15 foals. Three foals born from 2 different mares had presuckle antibodies at a titer ranging from 2,560 to 20,480. All 3 foals were born healthy. Two foals were born to the same dam that also gave birth to the weanling diagnosed with neuronal neosporosis in 2005. The third foal was born to a second mare with no previous foaling history at the farm. Seroconversion was documented in 10 foals and 9 mares over the 2-yr study. All blood and colostrum samples tested PCR negative for N. hughesi. Only 1 placenta collected in 2007 from the mare with the 2 congenitally infected foals tested PCR positive for N. hughesi. In conclusion, N. hughesi persisted in this population via endogenous transplacental infection.

Radiofrequency-induced thermal inactivation of Toxoplasma gondii oocysts in water.

Toxoplasma gondii, a ubiquitous parasitic protozoan, is emerging as an aquatic biological pollutant. Infections can result from drinking water contaminated with environmentally resistant oocysts. However, recommendations regarding water treatment for oocyst inactivation have not been established. In this study, the physical method of radiofrequency (RF) power was evaluated for its ability to inactivate T. gondii oocysts in water. Oocysts were exposed to various RF energy levels to induce 50, 55, 60, 70 and 80 degrees C temperatures maintained for 1 min. Post-treatment oocyst viability was determined by mouse bioassay with serology, immunohistochemistry and in vitro parasite isolation to confirm T. gondii infections in mice. None of the mice inoculated with oocysts treated with RF-induced temperatures of > or =60 degrees C in an initial experiment became infected; however, there was incomplete oocyst activation in subsequent experiments conducted under similar conditions. These results indicate that T. gondii oocysts may not always be inactivated when exposed to a minimum of 60 degrees C for 1 min. The impact of factors such as water heating time, cooling time and the volume of water treated must be considered when evaluating the efficacy of RF power for oocyst inactivation.

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi).

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.

Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters.

Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.

Evaluation of an indirect fluorescent antibody test (IFAT) for demonstration of antibodies to Toxoplasma gondii in the sea otter (Enhydra lutris).

An indirect fluorescent antibody test (IFAT) for detection of Toxoplasma gondii infection was validated using serum from 77 necropsied southern sea otters (Enhydra lutris nereis) whose T. gondii infection status was determined through immunohistochemistry and parasite isolation in cell culture. Twenty-eight otters (36%) were positive for T. gondii by immunohistochemistry or parasite isolation or both, whereas 49 (64%) were negative by both tests. At a cutoff of 1:320, combined values for IFAT sensitivity and specificity were maximized at 96.4 and 67.3%, respectively. The area under the receiver-operating characteristic curve for the IFAT was 0.84. A titer of 1:320 was used as cutoff when screening serum collected from live-sampled sea otters from California (n = 80), Washington (n = 21), and Alaska (n = 65) for T. gondii infection. Thirty-six percent (29 out of 80) of California sea otters (E. lutris nereis) and 38% (8 out of 21) of Washington sea otters (E. lutris kenyoni) were seropositive for T. gondii, compared with 0% (0 out of 65) of Alaskan sea otters (E. lutris kenyoni).

Coastal freshwater runoff is a risk factor for Toxoplasma gondii infection of southern sea otters (Enhydra lutris nereis).

The association among anthropogenic environmental disturbance, pathogen pollution and the emergence of infectious diseases in wildlife has been postulated, but not always well supported by epidemiologic data. Specific evidence of coastal contamination of the marine ecosystem with the zoonotic protozoan parasite, Toxoplasma gondii, and extensive infection of southern sea otters (Enhydra lutris nereis) along the California coast was documented by this study. To investigate the extent of exposure and factors contributing to the apparent emergence of T. gondii in southern sea otters, we compiled environmental, demographic and serological data from 223 live and dead sea otters examined between 1997 and 2001. The T. gondii seroprevalence was 42% (49/116) for live otters, and 62% (66/107) for dead otters. Demographic and environmental data were examined for associations with T. gondii seropositivity, with the ultimate goal of identifying spatial clusters and demographic and environmental risk factors for T. gondii infection. Spatial analysis revealed clusters of T. gondii-seropositive sea otters at two locations along the coast, and one site with lower than expected T. gondii seroprevalence. Risk factors that were positively associated with T. gondii seropositivity in logistic regression analysis included male gender, older age and otters sampled from the Morro Bay region of California. Most importantly, otters sampled near areas of maximal freshwater runoff were approximately three times more likely to be seropositive to T. gondii than otters sampled in areas of low flow. No association was found between seropositivity to T. gondii and human population density or exposure to sewage. This study provides evidence implicating land-based surface runoff as a source of T. gondii infection for marine mammals, specifically sea otters, and provides a convincing illustration of pathogen pollution in the marine ecosystem.

Immune responses in pregnant cattle and bovine fetuses following experimental infection with Neospora caninum.

Humoral and cell-mediated immune (CMI) responses [i.e. proliferative responses and gamma interferon (IFN-gamma) production], were elicited in five cows infected between 159 and 169 days of gestation by a combined intravenous-intramuscular inoculation of Neospora caninum tachyzoites. Analysis of antigen-specific immunoglobulin (IgG) subclasses revealed a predominant IgG2 response in two cows, a mixed IgG1-IgG2 response in two other cows and a predominant IgG1 response in one cow. No correlation was found between IgG2 titers and IFN-gamma levels. CD4-T cells were responsible for the CMI responses in peripheral blood mononuclear cells from three infected cows. All five fetuses removed from infected dams at week 9 post-infection (219-231 days of gestation) mounted strong Neospora-specific humoral responses and had a predominant IgG1 response, regardless of their ability to produce IFN-gamma. However, CMI responses were highly variable between fetuses. These data indicate the complexity of the immune mechanisms associated with Neospora infection in both the dams and their fetuses.

Isolation and characterization of two parasitic protozoa from a Pacific harbor seal (Phoca vitulina richardsi) with meningoencephalomyelitis.

Two species of protozoans were isolated from a harbor seal with fatal meninogoencephalitis. Serologic reactivity was detected to both Sarcocystis neurona and Toxoplasma gondii. Parasites associated with brain inflammation and necrosis reacted only with immunohistochemical stains utilizing polyclonal antisera raised against Sarcocystis neurona. However, 2 distinct parasites were observed in cell cultures derived from the seal's brain tissue. These parasites were separated by mouse passage and limiting dilution. Purified zoites from 1 isolate (HS1) reacted strongly with polyclonal antiserum to S. neurona and with the harbor seal's own serum (1:2,560 for each) on indirect immunofluorescent antibody tests (IFAT), but weakly to antisera to T. gondii and Neospora caninum (1:40). Zoites from the second isolate (HS2) reacted positively with T. gondii polyclonal antiserum (1:81,920) and with the harbor seal's own serum (1:640), but weakly to S. neurona and N. caninum antisera (1:80 or less). Amplification and sequence analysis of protozoal DNA encoding portions of the 18s ribosomal RNA (18s rDNA) and the adjacent first internal transcribed spacer (ITSI) were performed for both isolates, and resulting sequences were compared to those from similar protozoans. Based on molecular characterization, parasite morphology, serologic reactivity, histology, and immunohistochemistry, HS1 was indistinguishable from S. neurona, and HS2 was indistinguishable from T. gondii.

A POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation failed to prevent foetal infection in pregnant cattle following i.v./i.m. experimental tachyzoite challenge.

Cattle immunised with a POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation were previously shown to produce interferon (IFN)-gamma at levels similar to those of tachyzoite-infected cattle. In view of the critical role of IFN-gamma in resistance of mice to N. caninum infection, these results prompted us to test the POLYGEN-adjuvanted preparation in pregnant cattle to determine whether it will be able to prevent foetal infection following an experimental tachyzoite challenge. Seven heifers were immunised at 35 and 63 days of gestation with the POLYGEN-adjuvanted preparation, while five heifers were inoculated with POLYGEN alone at the same days of gestation. Four weeks later, all heifers were challenged with a combined i.v./i.m. inoculation of tachyzoites. The same challenge was given to seven unimmunized heifers at the same stage of gestation. An additional unimmunized heifer was inoculated with uninfected monolayer cell culture material. All challenged heifers, immunized and unimmunized, had infected foetuses. Immunized heifers developed both parasite-specific humoral and cellular immune responses, characterised by increased IFAT titres, a predominant IgG1 response, elevated lymphoproliferative response and IFN-gamma production. Following tachyzoite challenge, they developed an anamnestic humoral response and produced similar amounts of IgG1 and IgG2 antibodies, but did not have an anamnestic cellular immune response. The lack of anamnestic cellular immune response and/or the large i.v/i.m tachyzoite inoculum may have contributed to the failure of the preparation.

Immunogenicity of a killed whole Neospora caninum tachyzoite preparation formulated with different adjuvants.

A killed whole Neospora caninum tachyzoite preparation was formulated with various adjuvants and tested for its immunogenicity in cattle. The adjuvants used were: Havlogen, a polymer of acrylic acid cross-linked with polyallylsucrose; Polygen, a non-particulate copolymer; a mixture of Havlogen and Bay R-1005, which is a preparation of free base synthetic glycolipids; and Montanide ISA 773, a water-in-oil emulsion made with a mixture of metabolisable and mineral oils. Immune responses in immunised cattle were compared with those of cattle experimentally infected with culture-derived N. caninum tachyzoites. The overall mean serum IFAT titres were significantly higher (P < 0.05) in experimentally infected cattle compared with all immunised cattle. Nonetheless, the maximum antibody titres of the immunised cattle, which were obtained following the third immunisation, were within the range of titres previously described for naturally infected cattle. The overall mean serum IFAT titres were significantly higher (P < 0.05) in cattle immunised with the killed tachyzoite preparation formulated with Polygen and with the mixture of Havlogen and Bay R-1005, compared with cattle immunised with the Havlogen- and Montanide-based preparations. Two of the four adjuvant preparations were able to induce cell-mediated immune responses similar to those of the experimentally infected cattle. The Havlogen-adjuvanted tachyzoite preparation elicited N. caninum-specific proliferation of peripheral blood mononuclear cells statistically similar (P = 0.095) to that of the infected animals. Peripheral blood mononuclear cells from animals immunised with the Polygen-adjuvanted tachyzoite preparation produced interferon-gamma concentrations of similar magnitude (P = 0.17) to those from the infected animals. Polygen was one of two adjuvants that elicited the highest antibody responses, and was the only adjuvant that induced interferon-gamma levels similar to those of the infected heifers.

Description of a new Neospora species (Protozoa: Apicomplexa: Sarcocystidae).

Neospora hughesi n. sp. was isolated from the central nervous system tissue of an adult equine (Equus caballus) from California. The tachyzoites are crescent-shaped, approximately 2 x 5 microm (1.8-3.0 x 4.0-7.0 microm), with characteristic apical complex structures consisting of an anterior polar ring, conoid, numerous rhoptries filled with a uniform electron-dense material, and 22 microtubules extending posteriorly from the polar ring. Comparison of N. hughesi to canine and bovine Neospora caninum isolates showed phenotypic differences in immunoreactive proteins. Molecular analysis of the small subunit ribosomal RNA gene revealed no differences in the nucleotide sequence between N. hughesi and N. caninum isolates examined. However, the internal transcribed spacer I region revealed 7 nucleotide base differences between N. hughesi and N. caninum isolates (CN1 and BPA1) analyzed in this study. The existence of nucleotide base differences in the internal transcribed spacer regions suggests that this region may be a genetic marker for discriminating species within the genus Neospora. The ultrastructural, antigenic, and molecular data support distinction of N. hughesi as a new species, separate from N. caninum, the only recognized species in this genus.

A modified agglutination test for Neospora caninum: development, optimization, and comparison to the indirect fluorescent-antibody test and enzyme-linked immunosorbent assay.

Current serologic tests used to detect antibodies to Neospora caninum require species-specific secondary antibodies, limiting the number of species that can be tested. In order to examine a wide variety of animal species that may be infected with N. caninum, a modified direct agglutination test (N-MAT) similar to the Toxoplasma gondii modified direct agglutination test (T-MAT) was developed. This test measures the direct agglutination of parasites by N. caninum-specific antibodies in serum, thus eliminating the need for secondary host-specific anti-isotype sera. The N-MAT was compared to the indirect fluorescent-antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA) with a "gold standard" serum panel from species for which secondary antibodies were available (n = 547). All positive samples tested were from animals with histologically confirmed infections. Up to 16 different species were tested. The N-MAT gave a higher sensitivity (100%) and specificity (97%) than the ELISA (74 and 94%, respectively) and had a higher sensitivity but a lower specificity than the IFAT (98 and 99%, respectively). The reduced specificity of the N-MAT was due to false-positive reactions in testing fetal fluids with particulate matter or severely hemolyzed serum. Overall, the N-MAT proved to be highly sensitive and specific for both naturally and experimentally infected animals, highly reproducible between and within readers, easy to use on large sample sizes without requiring special equipment, and useful in testing serum from any species without modification.

Detection of Neospora from tissues of experimentally infected rhesus macaques by PCR and specific DNA probe hybridization.

Neospora is a newly recognized Toxoplasma-like cyst-forming coccidian parasite that causes abortion or congenital infections in naturally or experimentally infected animals. In this study, pregnant rhesus macaques were inoculated with culture-derived tachyzoites of a bovine Neospora isolate, and tissue samples from various major organs were collected from dams and fetuses for the detection of parasite DNA by using oligonucleotide primers COC-1 and COC-2 for PCR amplification of a conserved coccidial nuclear small-subunit rRNA gene sequence, and amplification products were confirmed by hybridization with a Neospora-specific DNA probe. PCR products were amplified from DNAs of different fetal monkey tissues, including brain, heart, lung, liver, spleen, skeletal muscle, skin, and placenta. In addition, Neospora DNA was amplified from the brain, heart, and lung tissues of infected rhesus macaque dams. The PCR and probe hybridization system may provide an effective method for the detection of Neospora infection in fetuses and dams from nonhuman primates and may be useful in determining the zoonotic potential of Neospora.

Detection of Neospora sp. from infected bovine tissues by PCR and probe hybridization.

Neospora sp. can cause fetal abortion or neurological disease in congenitally infected calves. Latent tissue stages in infected cows may contribute to vertical transmission of Neospora sp. from dam to offspring in multiple pregnancies. In this investigation, the polymerase chain reaction (PCR) and Neospora-specific assay were employed to detect Neospora sp. by amplification of nuclear small-subunit rRNA gene sequences in infected cattle tissues. Tissues from 11 cattle, including 6 experimentally and 2 naturally infected cows, 1 naturally infected newborn calf, and 2 uninfected control cows, were evaluated in this study. Neospora-specific PCR products were amplified from DNAs of different bovine tissues, including brain, spinal cord, heart, lung, kidney, diaphragm, skeletal muscle, and placenta, as well as amniotic fluid samples of infected cattle. The PCR-based amplification and probe hybridization system proved useful in assessing the location of tissue-stage parasites in naturally and experimentally infected cattle, even when Neospora sp. antibody titers fall below normal cut-off values by an indirect immunofluorescent antibody test.

Fatal hepatic sarcocystosis in two polar bears (Ursus maritimus).

Fatal hepatic sarcocystosis was diagnosed in 2 polar bears from a zoo in Anchorage, Alaska. Gross lesions were icterus and systemic petechiae. Marked microscopic lesions were detected only in the liver and included severe random necrotizing hepatitis with hemorrhage. Only asexual stages of an apicomplexan parasite were detected within hepatocytes, and rare extracellular zoites were seen in foci of necrosis. The parasite divided by endopolygeny, and occasionally merozoites formed rosettes around a central residual body. Ultrastructural features of the merozoites included a conoid and low numbers of micronemes at the apical pole, centrally located nuclei, and absence of rhoptries. The parasites failed to react with anti-Neospora sp., anti-Toxoplasma gondii, or anti-Sarcocystis neurona sera. The microscopic and ultrastructural morphology of the parasite are most compatible with an apicomplexan protozoan of the genus Sarcocystis. The life cycle of this parasite in bears is not known.

Evidence of vertical transmission of Neospora sp infection in dairy cattle.

OBJECTIVE: To determine whether heifers with naturally acquired congenital exposure to Neospora sp would transmit the infection to their offspring during gestation. DESIGN: Prospective cohort study. ANIMALS: Neonatal heifers on a dairy with a history of Neospora sp infections were selected for the study on the basis of their serum titers to Neospora sp, as determined by the use of indirect fluorescent antibody testing. Seropositive heifers (n = 25) had titers > or = 1:5,120 and seronegative heifers (25) had titers < or = 1:80. All heifers were raised and bred on the dairy, and samples were obtained from heifers and their calves at the time of calving. PROCEDURE: Blood samples were tested for Neospora sp antibodies. Histologic evaluations, Neospora sp immunohistochemical examinations, and protozoal culturing were performed on samples obtained from selected offspring (second-generation calves). RESULTS: Seropositive heifers gave birth to calves with titers > or = 1:1,280 to Neospora sp. All offspring from seropositive heifers that were necropsied had evidence of Neospora sp infection. All seronegative heifers and their offspring had titers < 1:80 to Neospora sp. CLINICAL IMPLICATIONS: Congenitally acquired Neospora sp infection can persist in clinically normal heifers and be transmitted transplacentally to their offspring. Vertical transmission can be a way by which neosporosis is maintained in herds.