Characterization of tissue distribution and histopathological lesions in <Emphasis Type="Italic">Neospora caninum</Emphasis> experimentally infected gerbils

Gerbils (Meriones unguiculatus) were inoculated intraperitoneally (i.p.) with Neospora caninum tachyzoites to examine parasite distribution and histological lesions at different time points over a 9-day period of infection. Gerbils were sacrificed 12 h post-infection (PI), then daily intervals up to day 9 PI. The parasite was detected by PCR assay targeting the Nc5 sequence of N. caninum. The parasite was not found in any organs until day 5 PI, however, from day 8 PI onwards, they were detected in all the organs examined as demonstrated by PCR. The first target organs in acute N. caninum infection were liver, spleen, and kidney, but not the blood as was expected. Histologic lesions were detected in the liver and spleen only, no lesions were found in other organs examined until the end of the experiment. Notably, the focal miliary hepatitis was observed in the liver of infected gerbils just after 1 day post-inoculation, whereas splenic lesions were not found until day 5 PI. These results reinforce the applicability of gerbils as a suitable model of acute neosporosis and provide new insights into the response of gerbils to N. caninum intraperitoneal infection.     

Seroprevalence of Neospora caninum and Toxoplasma gondii in camels (Camelus dromedarius) in Mashhad, Iran

One hundred twenty camels were blood-sampled and used to evaluate serological screening for Neospora caninum and Toxoplasma gondii infection by indirect fluorescent antibody test (IFAT) in Mashhad, Iran, during years 2004–2005. Of the 120 camels, antibodies to N. caninum were found in three in titers of 1:20 and in four in titers of 1:40 using whole N. caninum tachyzoites as IFAT slide (VMRD Inc., Pullman, WA 99163, USA). Antibodies to T. gondii were found in three camels in titers 1:20 and in two camels in titers 1:40 using whole T. gondii tachyzoites as IFAT slide (BIOGENE, Iran).     

Serologic study of anti-Neospora caninum antibodies in household dogs and dogs living in dairy and beef cattle farms in Tehran, Iran

A few studies have been done on the seroepidemiology of anti-Neospora caninum antibodies in dairy and beef cattle farms in Iran, which suggested the presence of N. caninum in these areas, but there is no published information directed on the presence or epidemiology of this organism in the dogs in Iran. To investigate anti-N. caninum antibodies in household dogs and dogs living in cattle farms, 100 blood samples were collected: 50 from dogs living in dairy and beef cattle farms and 50 from household dogs. Serum samples were screened for detection of anti-N. caninum IgG antibodies using indirect fluorescent antibody test (IFAT; ≥50). Antibodies were seen in 10 (20%) of 50 household dogs and in 23 (46%) of 50 farm dogs. There were significant statistical differences in seropositivity between these two groups (P = 0.005). The IFAT antibody titers were as follows: 1:50 in seven dogs, 1:100 in eight dogs, 1:200 in six dogs, 1:400 in seven dogs, 1:800 in three dogs, 1:1,600 in one dog, and 1:12,800 in one dog. There were no significant differences in seropositivity between males and females. The positive results were increasing with age, and positive results were significantly different in the age group of older than 2 years compared to the dogs of age group under 1 year (P = 0.000) and 1–2 years (P = 0.007). The results confirm the exposure of household and farm dogs to N. caninum in Tehran and the higher rate of exposure for the dogs of dairy and cattle farms around Tehran.     

Detection of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in water by an immunomagnetic separation method targeting the sporocysts

An immunomagnetic separation (IMS) method was developed to detect Toxoplasma gondii in fresh waters by using the monoclonal antibody 4B6 targeting the sporocyst wall of T. gondii, Hammondia hammondi, Hammondia heydorni, and Neospora caninum. Water concentrates obtained by filtering 10- to 20-l samples samples were spiked with Toxoplasma oocysts, sonicated to release the sporocysts, and analyzed by IMS-4B6. Mean sporocyst recoveries were 74.5 ± 5.3% in drinking water, 30.6 ± 2.4 and 37.1 ± 3.2% in surface waters, and 81.6 ± 2.1% in IMS buffer. Then, this IMS method was integrated in a multistep procedure (i.e., filtration, IMS, immunofluorescence and autofluorescence) to detect Toxoplasma in unspiked and spiked water samples (10–30 l) of various qualities. Sporocyst recoveries ranged from 14.4 to 44.7% in drinking water samples spiked with 1–10 oocysts/l, and from 17.8 to 32.5% in surface water samples spiked with 10 oocysts/l. Sporocysts were not detected in 25 unspiked water samples. A sporocyst-like structure was seen in one of these unspiked samples, but its coccidian nature could not be proved by three polymerase chain reaction (PCR) methods targeting sequences of coccidian small and large subunit rRNA genes and Toxoplasma repetitive elements. In conclusion, IMS-4B6 is relevant for the detection of Toxoplasma in water generating small concentrates (<1 ml). Due to 4B6 cross-reactions, a PCR would be useful to further characterize coccidian sporocysts found microscopically.     

Interaction of antibodies with Entamoeba histolytica trophozoites from experimental amebic liver abscess: an immunocytochemical study

Using immunocytochemical techniques, we studied the interaction of antibodies with Entamoeba histolytica trophozoites present during the development of amebic liver abscess. Hamsters were intrahepatically inoculated with HM1-IMSS axenic amebas and sacrificed at different days post-inoculation. IgG of rabbit anti-E. histolytica and IgG of rabbit anti-IgG of hamster were used, both labeled with peroxidase. With the rabbit anti-E. histolytica, all trophozoites present in hepatic lesions from 1–7 days post-inoculation were highly labeled. The IgG of rabbit anti-IgG of hamster intensively stained only those trophozoites present in lesions from 1–2 days post-inoculation. From day 3, the intensity and number of labeled trophozoites decreased progressively. The results suggest that the interaction between the amebas and the IgG of hamster is non-specific during the first 2 days. The absence of labeling in the chronic stages could be due to changes in the membrane antigens of the parasite or to alterations in the bloodstream around necrosis. Also, the anti-E. histolytica antibodies produced in the serum during the development of the hepatic disease are apparently incapable of reaching and interacting with the trophozoites present on the liver abscess. This can explain in part why antibodies do not have an important role in the defense of the host. Received: 26 September 1999 / Accepted: 24 November 1999     

Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Pará State, Amazon, Brazil

A cross-sectional study was conducted to determine the occurrence of anti-Toxoplasma gondii, anti-Neospora caninum, and anti- Leishmania chagasi antibodies in dogs of the state of Pará, Brazil. For this purpose, 129 blood samples were collected from dogs of different ages and gender. Samples of 72 dogs were collected from 39 rural properties from 19 municipalities, and 57 samples were from stray dogs, collected after captivity by the Center of Zoonosis Control from the municipality of Santarém. The sera were analyzed for anti-T. gondii and anti-N. caninum antibodies by indirect fluorescent antibody tests with cutoff values of 1:16 and 1:50, respectively. For the presence of L. chagasi antibodies, enzyme-linked immunosorbent assay was used and positive results were confirmed by immunochromatographic method using the recombinant antigen K39. Of the total of 129 dogs, 90 (69.8%) were positive for T. gondii, 16 (12.4%) for N. caninum, and 30 (23.3%) for L. chagasi. Antibodies for all three parasites were found simultaneously in seven dogs (5.4%), mostly in urban dogs (six of seven). No association was observed related to gender and location (urban or rural) of dogs and occurrence of N. caninum and T. gondii antibodies although, regarding L. chagasi, higher prevalence was found in females (P < 0.02) and in dogs from urban location (P < 0.001). From the 39 farms, in 30 (76.9%) at least one dog was positive for T. gondii or N. caninum or both. Higher occurrence of Leishmania antibodies was observed in N. caninum-negative dogs (P < 0.05).     

Dose dependency of prednisolone tertiary-butylacetate (PTBA) treatment on the establishment and site predilection of Echinococcus multilocularis in an alternative definitive host model using Mongolian gerbil (Meriones unguiculatus)

Mongolian gerbils (Meriones unguiculatus) were orally inoculated with 10,000 protoscoleces of Echinococcus multilocularis per head after being divided into five groups (A–E). Each group was dosed with prednisolone tertiary-butylacetate (PTBA) as follows: A, 0 mg; B, 0.5 mg; C, 2 mg; D, 5 mg; and E, 10 mg/head. All animals were injected subcutaneously with control solvent or PTBA every other day from 6 days pre- to 6 days post-infection. Autopsy was performed at 7 days post-infection. Doses of PTBA and the number of worms recovered showed a positive correlation (r=0.929, P < 0.0001). In groups A, B and C, the predilection site of the worms in the small intestine could not be determined, while in group D the worms were found more in the anterior part. In group E, the predilection site was the anterior part, followed by the middle and the posterior parts of the small intestine (Fisher's test: P < 0.01). The number of worms recovered from the anterior and the middle part of the small intestine also correlated positively with PTBA dose (anterior part: r=0.930, P < 0.0001, middle part: r=0.917, P < 0.0001). All groups of the PTBA-treated animals showed significant loss of weight compared to the non-treated animals (P < 0.01). Received: 26 September 1999 / Accepted: 24 November 1999     

Epidemiology of neosporosis in dairy cattle in Galicia (NW Spain)

This comprehensive study of neosporosis in dairy cattle in Galicia (NW Spain) included: (1) a comparative study of three serological techniques for detection of Neospora caninum antibodies (direct agglutination, enzyme-linked immunosorbent assay and indirect immunofluorescence); (2) a cross-sectional serological survey in which 276 herds and 5,196 animals were tested; (3) a study of N. caninum antibody dynamics; (4) the isolation of viable tachyzoites of N. caninum. Data were analysed to determine the risk factors associated with the infection. A total of 219 herds (79.3%) and 816 heads of cattle (15.7%) were found to be seropositive. Seropositivity was higher on farms with dogs than on farms without dogs, and there was a negative correlation between the size of the herds and seroprevalence. Co-infection with Toxoplasma gondii increased the risk of seropositivity. Cows infected with N. caninum were 5.3 times more likely to abort than non-infected cows. The dynamics study showed an increase in anti-N. caninum antibody titres during the third trimester of pregnancy. Viable tachyzoites were isolated from brain samples. These results indicate that the economic impact of N. caninum is high in Galicia, and therefore, the inclusion of control measures for neosporosis in the official control health programmes is strongly recommended.     

Efficacy of toltrazuril and ponazuril against experimental Neospora caninum infection in mice

Neosporosis is a disease affecting predominantly fetal development in cattle and dog hosts; and it may cause neuromuscular disfunction in infected new-born calves and pups. Predispositions – including, e.g. transient immunosuppression during pregnancy – may result in an increased dissemination of the parasite within the host or its offspring. Chemotherapeutic treatment of neosporosis may be an issue, provided that an appropriate drug is made available. In this respect, we describe the use of a mouse model for the evaluation of toltrazuril and ponazuril medication as a means of preventing parasite dissemination and subsequent formation of cerebral lesions. Toltrazuril- and ponazuril-treated mice were experimentally infected intraperitoneally (i.p.) with 2 × 10⁶ Neospora caninum tachyzoites. The infection was monitored at three levels: clinically, by assessing symptoms, histologically, by assessing the occurrence of cerebral lesions and parasites by immunohistochemistry, and on the molecular level, by detection of parasite DNA using PCR. Chemotherapy using either toltrazuril or ponazuril, both applied in a drinking-water formulation (20 mg toltrazuril or ponazuril kg⁻¹ body weight day⁻¹) completely prevented the formation of cerebral lesions in all treated animals, as assessed by immunohistochemistry. PCR analyses of these treated animals showed that DNA-detectability was reduced by 91% and 90% upon toltrazuril and ponazuril medication, respectively. Received: 6 August 2000 / Accepted: 7 August 2000     

Prevalence of antibodies to Neospora caninum in stray dogs of Urmia, Iran

To investigate anti-Neospora caninum antibodies in stray dogs living in Urmia city, 135 blood samples were collected. Serum samples were screened for detection of anti-N. caninum IgG antibodies using indirect fluorescent antibody test (IFAT; ≥50). Antibodies were seen in 36 (27%) of 135 dogs. The IFAT antibody titers were as follows: 1:50 in 16 dogs, 1:100 in ten dogs, 1:200 in six dogs, 1:400 in one dog, 1:800 in two dogs, and 1:1,600 in one dog. There were no significant differences in seroprevalence of Neospora infection between different genders (p > 0.05). The seropositive results were increased with age and the differences were statistically significant (p < 0.05). The results confirm the presence and exposure of stray dogs to N. caninum in Urmia city and the importance of this protozoan as a cause of disease in dogs of the region.     

Toltrazuril treatment of congenitally acquired <Emphasis Type="Italic">Neospora caninum</Emphasis> infection in newborn mice

C57BL/6 mice were infected with Neospora caninum tachyzoites during pregnancy, yielding a transplacental infection of developing fetuses. Subsequently, congenitally infected newborn mice were treated either once or three times with toltrazuril (or placebo) at a concentration of 31.25 mg compound per kg body weight. Both toltrazuril and placebo treatment had no negative effect on newborns, as noninfected treated pups developed normally without differences in mortality and morbidity to matching nontreated control animals. Already one application of toltrazuril was significantly (p < 0.01) able to delay the outbreak of neosporosis in newborn mice, when compared to placebo-treated infected controls. We found significantly higher proportion of surviving newborns in one-time-toltrazuril-treated and three-time-toltrazuril-treated groups (34% and 54%, respectively) when compared to one-time-placebo-treated and three-time-placebo-treated groups (14% and 30%, respectively). There was no significant difference (p = 0.2) in the proportion of surviving pups between one-time-toltrazuril and three-time-toltrazuril treatment. However, the number of diseased and Neospora-positive pups (46% and 47%, respectively) was markedly reduced after three-time-toltrazuril treatment compared to all other groups. Three-time-treatment also resulted in the highest antibody (IgG, IgG2a) response. Pharmacokinetic analyses using individual serum samples revealed that, although toltrazuril was absorbed and metabolized to toltrazuril sulfone by newborn mice, medicated animals exhibited an unexpected rapid turn-over (half-life time) of the compound. Toltrazuril and the metabolite were also found in brain tissues, indicating that passage of the blood–brain barrier occurred. In conclusion, we could show that three times treatment with toltrazuril had a high impact on the course of infection in congenitally N. caninum-infected newborn mice.     

Protective effect of intranasal immunization with Neospora caninum membrane antigens against murine neosporosis established through the gastrointestinal tract

Neospora caninum is an Apicomplexa parasite that in the last two decades was acknowledged as the main pathogenic agent responsible for economic losses in the cattle industry. In the present study, the effectiveness of intranasal immunization with N. caninum membrane antigens plus CpG adjuvant was assessed in a murine model of intragastrically established neosporosis. Immunized mice presented a lower parasitic burden in the brain on infection with 5 × 10⁷ tachyzoites, showing that significant protection was achieved by this immunization strategy. Intestinal IgA antibodies raised by immunization markedly agglutinated live N. caninum tachyzoites whereas previous opsonization with IgG antibodies purified from immunized mice sera reduced parasite survival within macrophage cells. Although an IgG1 : IgG2a ratio < 1 was detected in the immunized mice before and after infection, indicative of a predominant T helper type 1 immune response, no increased production of interferon‐γ was detected in the spleen or mesenteric lymph nodes of the immunized mice. Altogether, these results show that mucosal immunization with N. caninum membrane proteins plus CpG adjuvant protect against intragastrically established neosporosis and indicate that parasite‐specific mucosal and circulating antibodies have a protective role against this parasitic infection.     

Evidence of congenital transmission of Neospora caninum in naturally infected water buffalo (Bubalus bubalis) fetus from Brazil

The aim of this study was to determine the congenital infection by Neospora caninum in the water buffalo (Bubalus bubalis), a natural intermediate host. Nine pregnant water buffalos, raised under free-grazing condition, were slaughtered, and their fetuses were collected. Samples of brain and thoracic fluid were obtained from those fetuses, with gestational ages ranging from 2 to 5 months. The DNA of N. caninum was detected and identified in the brain of one of those fetuses, using two PCR assays, one directed to the Nc5 gene and the other, to the common toxoplasmatiid ITS1 sequence. The DNA fragments produced on PCR were sequenced, and N. caninum was confirmed in the samples. No antibodies to N. caninum were detected on any sample of thoracic fluid by immunofluorescent antibody test (IFAT < 25). This is the first confirmation of congenital transmission of N. caninum in water buffalos.     

First <Emphasis Type="Italic">in vitro</Emphasis> isolation of <Emphasis Type="Italic">Neospora caninum</Emphasis> from a naturally infected adult dairy cow in Slovakia

The impact of in vitro isolation and molecular characterisation of Neospora caninum as well as sequence analyses was studied. The brain homogenate of a naturally Neospora-infected dairy cow (positive in ELISA and Western blot) was intraperitoneally inoculated into Mongolian gerbils (Meriones unguiculatus). The brain of gerbils on day 60 post-inoculation was homogenized, and, after trypsin-digestion, cultured on Vero cells. Neospora-like tachyzoites were first observed after 77 days of cultivation. The parasite was confirmed by polymerase chain reaction (PCR) using Neospora-specific primers Np21 and Np6. The PCR product of the first Slovak isolate (NC-SKB1) was subsequently sequenced and published in GenBank under accession number GU300774. Sequencing and BLAST search identified the isolate as N. caninum.     

Seroprevalence of Neospora caninum infection in dogs in Chaharmahal-va-Bakhtiari Province, Iran

The aim of this study was to determine seroprevalence of Neospora caninum infection in dogs in Chaharmahal-va-Bakhtiari province in Iran. One hundred serum samples were collected from dogs of different sex, age, and breed. Collected serum samples were examined using an indirect fluorescent antibody test. Thirty-two of the examined samples showed detectable IgG antibodies in titers of 1:50. Dogs older than one year old and stray dogs had a higher seroprevalence than other dogs. No sex or breed predispositions to N. caninum infection were detected during this serological assay (P ≤ 0.05).     

Immunomodulatory effects in Litomosoides sigmodontis-infected Mastomys coucha

The levels of parasite-specific IgG₁ and IgG₂ antibodies and mitogen-induced and parasite-specific proliferative T-cell responses were determined in Litomosoides sigmodontis-infected Mastomys coucha throughout an observation period of 400 days post infection (p.i.). These were compared with the respective reactions in animals that had been immunized with intrauterine stages/microfilariae of the parasite and in animals that had been challenged after immunization as determined at up to 60 days after challenge. IgG₁ antibodies to adult antigen developed early and reached a plateau at 120 days p.i., whereas IgG₂ antibodies were not found before day 60 p.i., increased with rising parasitemia, reached a plateau at 200 days p.i., and, in some animals, even became the predominant IgG subclass. Proliferative responses of spleen lymphocytes to concanavalin A (Con A) and lipopolysaccharides (LPS), but not Con-A-induced interleukin 2 (IL-2) production, were found to be suppressed in infected animals during patency as compared with uninfected controls. Spleen cells of infected animals showed a weak proliferative reaction to male antigen but were unresponsive to female and microfilarial antigen during prepatency and early patency. Subsequently, when microfilaremia decreased (200 days p.i.), continuously increasing responses to all antigens were observed. Immunized M. coucha developed specific IgG₁ and IgG₂ antibodies, and their spleen cells showed strong proliferative responses to the three L. sigmodontis antigens. Challenge infections down-regulated the proliferative responses of spleen cells to filarial antigens as early as during the prepatent phase of the challenge infection but supported existing IgG₁ and IgG₂ responses. Received: 1 September 1999 / Accepted: 24 September 1999     

Light microscopic study on Eimeria species infecting Japanese quails reared in Saudi Arabian farms

Japanese quails Coturnix coturnix japonica reared in economic farms were individually investigated for coccidian infections. The results indicated the absence of infections in birds younger than 1 month. An Eimeria infection rate of up to 80% was detected in birds 7–9 weeks old with a general infection rate of 29%. The infection rate decreased to 21.42% in birds older than 10 weeks. Morphometric characteristics of freshly shed, unsporulated oocysts were taken. These oocysts appeared pale yellow in color, were oval to subspherical in shape being limited by a bilayered oocyst wall of 1.2 μm. The unsporulated oocysts measured 17.73 ± 12.92 × 12.79 ± 1.69 μm (mean of 100) and possessed a polar granule, a micropyle and an oocyst residuum. The sporulation took 72 h and resulted in the formation of four elongated sporocysts containing two sporozoites, in addition to a stieda body and a sporocyst residuum. The life cycle of this Eimeria species was followed in experimentally infected quails. Three asexual generations (at 60, 78, and 96 h p.i.) were detected in the epithelium of the small intestine before the sexual cycle started at 84 h p.i. The prepatent period was 5 days, while the patent period covered 6–7 days. Besides this well-defined species, another Eimeria species occurred, the oocysts of which were excreted in low numbers and were characterized by the absence of a micropyle and an oocyst residuum. These oocysts measured 15.73 ± 2.22 × 14.18 ± 1.89 μm (mean of 100) and sporulated already within 60 h.     

Development of high numbers of blood trypomastigotes of Trypanosoma cruzi in nude rats

The development and pathogenicity of a Trypanosoma cruzi strain (“Chile 5”) of low virulence were studied after infection of nude rats with different doses of blood trypomastigotes (10–10⁷ parasites/rat). Peak parasitemias were correlated with the infection dose, which also influenced the mean survival times (26–36 days post-infection). Within 26 or 27 days, a subcutaneous injection of 10⁷ blood trypomastigotes developed to about 8–20 × 10⁷ parasites/ml. Received: 12 September 2000 / Accepted: 12 October 2000     

Prevalence of antibodies against Neospora caninum in dogs from urban areas in Central Poland

Neospora caninum is a protozoan parasite which causes abortion in cattle as well as reproduction problems and neurological disorders in dogs. To assess the prevalence of the parasite in urban dogs in the Mazovian Voivodeship, Central Poland, serum samples from 257 dogs were analyzed for the presence of specific IgG antibodies. The examined dogs visited three private veterinary clinics located in Warsaw due to control tests, vaccinations, or other reasons not directly connected with neosporosis. Using ELISA and Western blot, antibodies against the parasite were detected in 56 out of 257 dogs, giving a prevalence of 21.7%. A greater prevalence was observed in female dogs than in males, 28% and 17.3%, respectively, and the differences were statistically significant (p < 0.05). There were no significant differences in seroprevalence of Neospora infection within the age groups (p > 0.05). This study indicates the presence of N. caninum in the Mazovian Voivodeship, in dogs which live in urban areas and exposure of these dogs to the parasite. The fact that seropositive dogs had no contact with cattle confirms the important role of dogs in the parasite’s epidemiology.     

<Emphasis Type="Italic">Eimeria biarmicus</Emphasis> sp.n. (Apicomplexa: Eimeriidae) infecting falcons from the genus <Emphasis Type="Italic">Falco</Emphasis> in Saudi Arabia

The oocysts of Eimeria biarmicus sp. n. were described from the feces of the lanner falcon, Falco biarmicus, collected from the falcon market in Riyadh City, Saudi Arabia. The prevalence of infection was 5% (2/40). The majority of the oocysts examined had completed sporulation within 84 h at 24 ± 2°C. Sporulated oocysts are ovoid in shape, measuring 22.4 × 17.9 (20.5–24.7 × 15.8–18.5) μm; shape index (L/W) is 1.25 (1.14–1.36) μm. The oocyst wall is smooth and bi-layered. Micropyle and oocyst residuum are absent. A polar granule is present, consisting of 2–4 globules. Sporocysts are ovoid, 10.1 × 6.1 (9.4–11.2 × 5.4–6.8) μm; with a smooth single-layered wall and a minute Stieda body, but there is no substieda body. The sporocyst residuum consists of numerous small granules. Sporozoites are comma shaped, each contains two refractile bodies. E. biarmicus sp. n. is the second eimerian species described from F. biarmicus.