Cryptosporidium parvum gp60 subtypes in diarrheic lambs and goat kids from Israel.

Cryptosporidium parvum is the second-most prevalent Cryptosporidium species that infects humans worldwide. In European countries, it is the most prevalent species in sheep, suggesting that these animals are a source of zoonotic infection. Preweaned lambs and goats are particularly susceptible to infection by the parasite and may suffer from severe diarrhea whilst excreting large quantities of infectious oocysts. Fifty fecal samples from preweaned lambs and goats with diarrhea from 35 farms across Israel, found to be Cryptosporidium-positive by microscopy, were tested by PCR and sequence analyses to determine the infective species and subtypes. Cryptosporidium parvum DNA was detected in most samples from both lambs and goats (46/50). Cryptosporidium xiaoi DNA was detected in three samples from kids, with co-infection detected in a single sample. Eleven different C. parvum subtypes were found, 10 in lambs and 5 in goats. All subtypes were from the IIa and IId subtype families, with subtypes IIdA20G1 and IIaA15G2R1 being the most prevalent and widespread. These subtypes were previously found in calves and humans in Israel and are considered the most prevalent C. parvum subtypes in small ruminants globally. These results underline the zoonotic potential of C. parvum from small ruminants and the high subtype diversity compared to previous reports from other Middle Eastern countries. In addition, this is the first report of C. xiaoi in Israel.

Cryptosporidium parvum subtypes from diarrheic dairy calves in Israel.

Cryptosporidium are protozoan parasites with worldwide distribution, infecting a wide range of terrestrial and aquatic animals, as well as humans. Cryptosporidium parvum is the most important zoonotic species and is the primary cause of cryptosporidiosis in preweaned calves, a highly prevalent, economically important disease. Extensive subtyping of C. parvum from infected humans and animals has expanded current understanding of the parasites' epidemiology. Israel has a highly developed dairy sector with intensive, zero-grazing operations. While C. parvum has been found in dairy calves throughout the country, and subtype data from human patients have also been published, subtype data from animals, and in particular preweaned ruminants, are lacking. We carried out an initial study of Cryptosporidium species and subtypes from preweaned diarrheic calves. Cryptosporidium species were determined in 71 fecal samples from 43 different dairy farms using 18S rRNA PCR, and subtyping of C. parvum based on the 60-kDa glycoprotein (gp60) sequences was done on one sample per farm. C. parvum was the only species found, with eight different subtypes belonging to the zoonotic IIa and IId families. Subtype IIaA15G2R1 was the most prevalent and widespread, found in 50% of the farms over a wide geographical distribution. Our results confirm the presence of subtypes IIaA15G2R1 and IIdA20G1, which were previously found in human patients in Israel, also in Israeli calves. In addition, subtype IIaA12G1R1 is reported here for the first time in an animal. These findings demonstrate the value of monitoring C. parvum subtypes in animal samples, and suggest that the role of calves as well as other young ruminants in the transmission of zoonotic cryptosporidiosis in Israel should be further studied.

Establishing Babesia bovis-Free Tick Colony Following Treatment of the Host with Diminazene Aceturate (Berenil).

Babesia bovis is a widely-spread tick-borne hemoparasite of cattle with major economic and animal welfare consequences. Rhipicephalus (Boophilus) annulatus is a one-host tick which transmits bovine babesiosis in the Middle East and Africa. Laboratory rearing of ixodid ticks is essential for the investigation on ticks or tick-borne diseases. Establishing a tick colony in the laboratory usually originates from ticks harvested in the field, which may be naturally infected with various pathogens. This especially applies to carriage of B. bovis as it is highly prevalent in endemic areas and is transmitted transovarially in ticks. Here, we describe the use of diminazene aceturate (Berenil) in order to establish laboratory colonies of Babesia-free R. annulatus, from ticks collected in the field. Ticks collected in the field were kept until oviposition and hatched larvae were introduced to naïve calves, which led to infection of the calves with B. bovis. Calves were then treated with diminazene aceturate several times until the engorged ticks dropped. The eggs and larvae collected from these ticks were parasite-free, as demonstrated both by infection of splenectomized calves and by PCR. This suggested protocol is a useful tool to create parasite-free tick colony and may, theoretically, also be beneficial to reduce parasite circulation in the field, although not recommended, as resistance to diamenizene aceturate might develop.

Experimental Infection of Calves with Transfected Attenuated Babesia bovis Expressing the Rhipicephalus microplus Bm86 Antigen and eGFP Marker: Preliminary Studies towards a Dual Anti-Tick/Babesia Vaccine.

Bovine babesiosis, caused by Babesia bovis and B. bigemina, is a major tick-borne disease of cattle with global economic impact. The disease can be prevented using integrated control measures including attenuated Babesia vaccines, babesicidal drugs, and tick control approaches. Vaccination of cattle with the Rhipicephalus microplus Bm86-based recombinant vaccine reduces the fitness of R. microplus and R. annulatus, but several booster inoculations are required to maintain protection. Herein, we generated a stable transfected strain of B. bovis expressing an enhanced GFP (eGFP) and a chimeric version of Bm86 (B. bovis/Bm86/eGFP). The eGFP was expressed in the parasite cytoplasm, whereas Bm86 was displayed on the surface of merozoites. Three splenectomized calves experimentally infected with B. bovis/Bm86/eGFP showed mild signs of acute disease and developed long-lasting antibody responses to B. bovis and native Bm86. No evidence of sequestration of parasites in the cerebral capillaries was found upon postmortem analysis, confirming attenuation of the strain. This is the first report of transfected B. bovis expressing the tick antigen Bm86 on the merozoite surface that elicits an antibody response to native Bm86. These results represent a proof of concept for a novel live, attenuated, tagged dual-vaccine approach to attempt simultaneous control of babesiosis and tick infestation.

High Exposure to Toxoplasma gondii and Neospora Spp. in Donkeys in Israel: Serological Survey and Case Reports.

Toxoplasma gondii and Neospora spp. are closely related cyst-forming coccidian parasites, which infect various animal species and have considerable zoonotic and economic implications, respectively. Both parasites are endemic in Israel and have been reported to infect wild and domestic animals. This study was conceived to evaluate the serologic exposure of donkeys to these parasites. Serum samples were collected from 98 donkeys. Half of them (n = 49) were from animal shelters in Israel, and the rest (n = 49) were working donkeys from the Palestinian Authority. The donkeys were screened for the presence of anti-Toxoplasma and anti-Neospora antibodies by immunofluorescence antibody tests (IFATs). The seroprevalence of T. gondii and Neospora spp. was 94% and 70%, respectively, and 69% of the donkeys were exposed to both parasites. In addition, N. caninum tissue cysts were documented in two donkeys during post-mortem examination. This is the first report of the exposure of donkeys to T. gondii and Neospora spp. in the area. The high prevalence found in this study suggests that donkeys may have a role in the maintenance of these parasites in the area, thus serving as a source of infection for the definitive hosts.

Neospora caninum: Chronic and congenital infection in consecutive pregnancies of mice.

Neospora caninum, the causative agent of bovine neosporosis is the major cause of abortion in cattle worldwide. The principal route of transmission is via in utero infection of the offspring. Congenitally-infected dams remain persistently infected for life and might undergo abortions in consecutive pregnancies. In the present study, the effect of N. caninum in chronic and congenital infection was examined. CD1 mice were infected intra-peritoneally with live tachyzoites of the NcIs491 isolate, while non-infected mice served as a control. There were no clinical signs of infection observed following inoculation, but high titers of specific anti- N. caninum antibodies were detected. A month after infection, when chronic-infection was established, mice were mated. Fertility, litter size and mortality rate were monitored within two generations of four consecutive pregnancies. During a nine months period of the study all females maintained high level of antibodies, while the non- infected control mice remained seronegative. There was no difference in the fertility rate of the dams, or in the litter size of infected and control mice. Mortality of offspring of the first and second generations of the infected dams was observed within the two first weeks of life. The vertical transmission was analyzed by PCR assay of offspring brains. PCR positive results were found in all 13 litters of the first generation tested during four consecutive pregnancies. The rate of vertical transmission slightly decreased in successive pregnancies being 74.2%, 59.5%, 48.1% and 40% for the first to fourth pregnancies respectively. In the second generation 21 out of 28 litters were found positive and the overall rate of vertical transmission was 28.5%. In chronically and congenitally infected dams N. caninum infection was maintained during all successive pregnancies for about 9 months. The results show that CD-1 outbred mice are a suitable model for studying chronic and congenital neosporosis.