Patricia A. Conrad

Neosporosis in cattle

During the past decade, Neospora caninum infection has emerged as an important reproductive disease in cattle throughout the world. Abortion, occurring during the middle of gestation, is the primary clinical sign of the infection in cattle. Surveys in several countries from three continents have identified N. caninum infection as the major diagnosed cause of bovine abortion. Both endemic and epidemic patterns of abortion may occur in herds. An important feature of this disease is that the protozoan parasite is maintained in cattle as a chronic infection which can be passed on to the fetus during pregnancy. Two methods for the transmission of the infection in cattle have been proposed and are the subject of current investigations. Horizontal transmission utilizes a two-host life cycle whereby the cow is infected from ingestion of coccidial oocyst stages shed by the definitive host. Experimental infections have confirmed that the dog is a definitive host for the parasite. There is epidemiological evidence that the dog has a role in the prevalence of the infection but, as yet, no confirmation that the dog is the source for natural infections in cattle. Vertical transplacental transmission of the infection is an important route of infection in many herds. Vertical transmission occurs because fetal infection frequently does not result in abortion but rather the fetus survives to be a persistently infected animal. A heifer calf that is born congenitally infected is capable of transmitting the infection to the next generation when she becomes pregnant, thus maintaining the infection in the herd. The clinical outcome of transplacental fetal infection with N. caninum is likely determined by maternal and fetal immune responses which involve humoral, and most importantly, cell-mediated immune factors. The diagnosis of the infection is assisted through histopathology and immunohistochemical examination of aborted fetuses and serologic testing of cattle for evidence of infection. Several types of serologic tests, based on the use of culture-derived organisms or recombinant N. caninum antigens are available. There are no proven control methods for the prevention or treatment of neosporosis. Suggested control measures focus on programs to reduce the number of congenitally infected animals retained in the herd and to minimize the opportunity for postnatal transmission from the environment.

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.

Human babesiosis : an emerging tick-borne disease

Human babesiosis is an important emerging tick-borne disease. Babesia divergens, a parasite of cattle, has been implicated as the most common agent of human babesiosis in Europe, causing severe disease in splenectomized individuals. In the US, Babesia microti, a babesial parasite of small mammals, has been the cause of over 300 cases of human babesiosis since 1969, resulting in mild to severe disease, even in non-splenectomised patients. Changing ecology has contributed greatly to the increase and expansion of human babesiosis in the US. A relatively recently described babesial parasite, the WA1-type, has been shown to be the causative agent in seven human cases in the western US. This parasite is closely related to babesial parasites isolated from large wild ungulates in California. Like B. microti, WA1-type parasites cause mild to severe disease and the immunopathogenesis of these parasites is distinctly different from each other in experimental infections of hamsters and mice. A B. divergens-like parasite was also identified as the cause of a fatal human babesiosis case in Missouri. Isolated cases of human babesisosis have been described in Africa and Mexico, but the causative parasites were not well characterized. Standard diagnostic techniques for human infection, such as examination of Giemsa-stained thin blood smears and serology, have been complemented with molecular techniques, such as PCR. Current treatment for babesiosis is focused on a regimen of clindamycin and quinine, although new drugs have shown promise. Prevention of infection relies on self-monitoring for the presence of ticks and, in some locations, targeted application of pesticides to decrease tick abundance. Identification of human infection with Babesia spp. will probably increase as physicians and the public become more aware of the disease, as people live and recreate in rural tick-infested areas, and as the numbers of immunocompromised individuals increase.

Detection of Serum Antibody Responses in Cattle with Natural or Experimental Neospora Infections

Parasite-specific antibody responses were detected using an indirect fluorescent antibody (IFA) test in cattle that were naturally or experimentally infected with Neospora parasites. The test was developed using Neospora tachyzoites isolated from an aborted bovine fetus and grown in bovine cell cultures (isolate BPA1). In all cases, infections were confirmed by the identification of Neospora tachyzoites and/or bradyzoite cysts in fetal or calf tissues using an immunoperoxidase test procedure. Fifty-five naturally infected cows that aborted Neospora-infected fetuses had titers of 320-5,120 at the time of abortion. The titer of 6 cows that were serologically monitored over a prolonged period decreased to 160–640 within 150 days after they aborted infected fetuses. Two of the cows showed an increase in their Neospora titers during their subsequent pregnancy, and they gave birth to congenitally infected calves that had precolostral titers of 10,240-20,480. Postcolostral titers of these calves and of 4 other calves with congenital Neospora infections were all 25,120, whereas calves with no detectable parasites had titers ≤ 160. Two pregnant heifers that were experimentally infected with the BPA1 isolate at approximately 120 days gestation seroconverted to Neospora antigens within 9 days and developed peak titers of 5,120 and 20,480 within 32 days of infection. The fetus taken by caesarian section 32 days postinfection from 1 heifer and the full-term calf born to the other had Neospora titers of 640 and 10,240, respectively. Nine cows that aborted uninfected fetuses and 61 adult cattle maintained under pasture or feedlot conditions, where risk of exposure to Neospora was considered to be low, had titers ≤ 320. Some of the feedlot cattle tested had serologic reactivity that was restricted to antigens at the apical end of both Neospora and Toxoplasma gondii tachyzoites. This type of reactivity, which may result from serologic cross-reactivity between conserved apical complex antigens of closely related sporozoan parasites, differed from the whole parasite fluorescence that was observed with sera from Neospora-infected animals. The significance of these results and the potential application of the IFA test for the diagnosis of Neospora infections in cattle are discussed.

Immune responses to Neospora caninum and prospects for vaccination

Developing an effective vaccine against neosporosis presents several interesting challenges. The parasite is spread efficiently from mother to foetus over several generations, and naturally infected cattle do not appear to develop adequate protective immunity. Modulation of the immune response during pregnancy favours parasite survival and multiplication. However, induction of pro-inflammatory responses that are thought to be protective against Neospora caninum would be detrimental to the pregnancy. So, is vaccination a feasible option to control the disease? This article discusses some of these issues and reports on the progress towards a vaccine for neosporosis.

PATTERNS OF MORTALITY IN SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS) FROM 1998–2001

Detailed postmortem examination of southern sea otters (Enhydra lutris nereis) found along the California (USA) coast has provided an exceptional opportunity to understand factors influencing survival in this threatened marine mammal species. In order to evaluate recent trends in causes of mortality, the demographic and geographic distribution of causes of death in freshly deceased beachcast sea otters necropsied from 1998–2001 were evaluated. Protozoal encephalitis, acanthocephalan-related disease, shark attack, and cardiac disease were identified as common causes of death in sea otters examined. While infection with acanthocephalan parasites was more likely to cause death in juvenile otters, Toxoplasma gondii encephalitis, shark attack, and cardiac disease were more common in prime-aged adult otters. Cardiac disease is a newly recognized cause of mortality in sea otters and T. gondii encephalitis was significantly associated with this condition. Otters with fatal shark bites were over three times more likely to have pre-existing T. gondii encephalitis suggesting that shark attack, which is a long-recognized source of mortality in otters, may be coupled with a recently recognized disease in otters. Spatial clusters of cause-specific mortality were detected for T. gondii encephalitis (in Estero Bay), acanthocephalan peritonitis (in southern Monterey Bay), and shark attack (from Santa Cruz to Point Año Nuevo). Diseases caused by parasites, bacteria, or fungi and diseases without a specified etiology were the primary cause of death in 63.8% of otters examined. Parasitic disease alone caused death in 38.1% of otters examined. This pattern of mortality, observed predominantly in juvenile and prime-aged adult southern sea otters, has negative implications for the overall health and recovery of this population.

Infection with a Babesia-Like Organism in Northern California

BACKGROUND Human babesiosis is a tick-transmitted zoonosis associated with two protozoa of the family Piroplasmorida: Babesia microti (in the United States) and B. divergens (in Europe). Recently, infection with an unusual babesia-like piroplasm (designated WA1) was described in a patient from Washington State. We studied four patients in California who were identified as being infected with a similar protozoal parasite. All four patients had undergone splenectomy, three because of trauma and one because of Hodgkins disease. Two of the patients had complicated courses, and one died. METHODS Piroplasm-specific nuclear small-subunit ribosomal DNA was recovered from the blood of the four patients by amplification with the polymerase chain reaction. The genetic sequences were compared with those of other known piroplasm species. Indirect immunofluorescent-antibody testing of serum from the four patients and from other potentially exposed persons was performed with WA1 and babesia antigens. RESULTS Genetic sequence analysis showed that the organisms from all four patients were nearly identical. Phylogenic analysis showed that this strain is more closely related to a known canine pathogen (B. gibsoni) and to theileria species than to some members of the genus babesia. Serum from three of the patients was reactive to WA1 but not to B. microti antigen. Serologic testing showed WA1-antibody seroprevalence rates of 16 percent (8 of 51 persons at risk) and 3.5 percent (4 of 115) in two geographically distinct areas of northern California. CONCLUSIONS A newly identified babesia-like organism causes infections in humans in the western United States. The clinical spectrum associated with infection with this protozoan ranges from asymptomatic infection or influenza-like illness to fulminant, fatal disease.

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.

Neospora-like Protozoal Infections Associated with Bovine Abortions

Eighty bovine fetuses with presumed protozoal infections from a previous 2-year retrospective study were examined by immunohistochemistry using antisera against Neospora caninum. In 66 (83%) of the fetuses, protozoa were found that reacted positively with anti-N. caninum sera. In three (4%) additional fetuses, protozoa identified as Sarcocystis species did not react, and in two fetuses (3%) single protozoal clusters were found only in hematoxylin and cosin-stained slides. A group of 20 fetuses were chosen for further evaluation. They included 14 fetuses from the first group of 80 fetuses plus six additional fetuses that had large numbers of protozoa in the fetal brain. The 20 fetuses were examined immunohistochemically with antisera to N. caninum, Hammondia hammondi, and Toxoplasma gondii. Protozoa from 3/20 fetuses, identified as Sarcocystis species, failed to react with any antisera. In 16/20 fetuses protozoa reacted positively to antisera against N. caninum, and in most cases reacted to H. hammondi, and weakly to one or more of the antisera against T. gondii. Thickwalled protozoal tissue cysts were found in the brain of four of these 16 fetuses by transmissiosn electron microscopy. The cyst wall morphology was comparable to N. caninum. The results suggested that a single protozoal parasite of unknown identity was responsible for most of the bovine abortions. By immunohistochemistry, the unknown protozoon reacted most strongly and consistently to N. caninum antisera, but was antigenically distinct from N. caninum. Ultrastructurally, tissue cysts found in four fetuses most closely resembled Neospora caninum.

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.