Development and application of a standardized assay for chemical disinfection of coccidia oocysts.

The development and application of a standardized model for testing of anticoccidial disinfectants are described. Due to its economic impact, tenacity of oocysts, and reproducibility of the course of infection Eimeria tenella has been chosen as test organism. Oocysts of the Houghton strain were more susceptible to disinfection with 4% TP4 (Preventol) than oocysts of a field isolate (FI 292/1) as determined by sporulation inhibition and lysis. Scoring of intestinal lesions and of oocyst numbers in mucosal scrapings in chicken infected with various doses of oocysts were found unsuitable for assessment of disinfectants. Because strain differences were observed only Houghton strain oocysts were applied for further testing. Guidelines for standardized in vivo testing of disinfectants have been stipulated by the German Veterinary Society (DVG) on the basis of these studies. When applied for testing of Neopredisan (NP) in two separate laboratories similar results were obtained. Inhibitory activity (IA; proportion of inactivated oocysts) of 92.9 and 90.6% were calculated for 3% NP and of 95.2 and 96.8% for 4% NP after treatment with the disinfectant over 120 min. According to the guidelines IA of at least 95% is required for certification of sufficient disinfecting efficacy by the DVG.

Seroepidemiologic studies on Babesia equi and Babesia caballi infections in Brazil.

Horses from six stud farms representing the most frequent types of horse breeding in Brazil were tested for Babesia antibodies by the IFA test. The farms are located at the tropic of Capricorn at an altitude of 472-715 m where temperatures below 0 degrees C may occur. Horses of conventional stud farms were infested with Dermacentor nitens, Amblyomma cajennense, and Boophilus microplus. Infestation with Bo. microplus was associated with direct or indirect contact of horses with cattle, and was not detected at professional stud farms. At one large professional stud farm, only D. nitens was observed. Prevalence of Babesia equi correlated positively (p<0.001) with contact of pastured horses to cattle (67.1% versus 17.5%). The IFA test was validated using sera from 92 non-infected horses and from 18 ponies infected experimentally with the USDA strains of B. equi or B. caballi or with the Brazilian isolates from the study area. Differences in test results obtained using antigens from USDA strains or Brazilian isolates were not significant. The specificity was 100% except for the IFA test using Brazilian antigen of B. caballi (98%). The sensitivity was 100% except for the IFA test using the USDA antigen of B. caballi. Accuracy of the tests ranged from 98 to 100%, and predictive values from 99 to 100%. Only 59% (132/224) of sera, positive by the IFA test for B. equi, tested positive by CF test, and 45% (232/515) of sera, positive by the IFA test for B. caballi, also tested positive by CF test. In all, 740 field sera from 140 horses, including 63 mares and their foals, were tested. Prevalence and incidences of B. caballi infections were significantly higher than those of B. equi infections: 49.2% (31/63) of the mares were infected with B. equi, but 79.4% (50/63) with B. caballi; 36% (18/50) of the foals became infected with B. equi within 12 months, but 100% (50/50) with B. caballi within 10 months. Maternal antibodies against B. equi and B. caballi in foals were 44 (22/50) and 68% (34/50), respectively. Titers persisted for 1-5 months for B. equi and 1-4 months for B. caballi.

Tick-borne diseases of sheep and goats caused by Babesia, Theileria or Anaplasma spp.

A review is given on the Babesia, Theileria, and Anaplasma species infecting sheep and goats. B. ovis is the most important disease agent. It is transmitted by Rhipicephalus bursa, R. turanicus, Hyalomma anatolicum excavatum, and probably by R. evertsi evertsi B. ovis is widely spread in southern Europe, the Middle East, and central Asia. Its geographical distribution in South and East Asia and in Africa is widely unknown. B. motasi obviously represents several nosodemes in separate regions. It is not pathogenic for intact sheep in northern Europe, whereas it is probably more pathogenic than B. ovis in India and northern Africa. The known vectors of B. motasi are Haemaphysalis punctata and R. bursa. Theileria hirci is transmitted by H. a. anatolicum but occurs outside the distribution area of this tick. Malignant theileriosis of sheep and goats is an important disease in Iraq, Iran, and India. An attenuated macroschizont vaccine is successfully being used in Iran. Anaplasma ovis is transmitted by R. bursa and probably other ticks in the Old World and by Dermacentor andersoni in the New World. A. ovis is widely spread in the Old World. Outbreaks occur only under extreme conditions. The identity of the tick-borne disease agents of sheep and goats and of their vector ticks is uncertain in many regions of the Old and the New World.

Epidemiological aspects of equine babesioses in a herd of horses in Brazil.

Epidemiological studies of Babesia equi and B. caballi were undertaken in a herd of 120 pastured horses in Rio de Janeiro, Brazil. The area where the horses were held was shown to be highly endemic for both Babesia spp., i.e. the prevalence of B. equi antibodies in horses aged 6 months or older ranged from 90.6% to 100% as determined by the immunofluorescence antibody (IFA) test, and the prevalence of B. caballi antibodies as determined by Western blot ranged from 59.4% to 65.5%. From the herd, 20 foals and their dams were selected to estimate the degree of tick infestation and the foals were bled at monthly intervals to determine the incidence of antibodies to B. equi and B. caballi. The incidence of B. equi was 100% by about 127 days of age as determined by IFA of B. caballi was 100% by about 150 days of age as determined by Western blot. Tick infestation of the horses estimated by using a semiquantitative key ranged from at least five ticks on every horse to more than 100 ticks on many horses throughout the year. Except for three Boophilus microplus female ticks, they were identified as Amblyomma cajennense and Anocentor nitens. A. cajennense had one generation per year, whereas An. nitens had three. Kinetes of B. caballi were detected in the haemolymph of two of 68 An. nitens female ticks and in the ovary and eggs of one of these, suggesting that this tick is a significant vector of B. caballi.(ABSTRACT TRUNCATED AT 250 WORDS)

Current state and future trends in the diagnosis of babesiosis.

An overview is given of the currently available methods to diagnose babesiosis in livestock. Microscopic techniques are still the only appropriate techniques to diagnose acute disease. Thin or thick blood films stained with Giemsa's stain are sufficient. The sensitivity ranges from 10(-5) to 10(-6), i.e. one parasite per 10(5)-10(6) erythrocytes can be detected. Thick films stained with acridine orange (sensitivity approximately 10(-7)) and the Quantitative Buffy Coat (QBC) analysis tube system (sensitivity approximately 10(-7)-10(-8)) are applicable for diagnosis in the laboratory. DNA probes are very specific tools to identify haemoparasites in organs post mortem and in ticks. For the identification of carrier animals the sensitivity (approximately 10(-5)-10(-6)) is generally not sufficient. For the latter the polymerase chain reaction (PCR) technique is a very powerful tool (sensitivity approximately 10(-9)). Many different serodiagnostic tests have been described; however, the immunofluorescence antibody test is the most widely used, while the enzyme-linked immunosorbent assay (ELISA) is the test system which holds the greatest promise for the future. Thus far, improvements to the ELISA have been limited as the quality of antigen preparations made from infected blood is generally poor with a few exceptions (Babesia bovis, Babesia caballi). Potentially, most of the problems associated with crude antigens can be overcome by the production of recombinant antigens. Several ELISAs based on highly defined recombinant antigens have been described and show promise. None of these tests has been validated to the extent that it could be applied globally. Future research requirements as well as the need for coordination of the research effort and collaboration between institutions involved in the diagnosis of babesiosis are discussed.

Host specificity of cloned Spironucleus sp. originating from the European hamster.

In vivo experiments with a clone of the intestinal flagellate Spironucleus sp. originating from the laboratory European hamster (Cricetus cricetus), and the comparison with a spontaneous infection from laboratory bred European hamsters suggest high specificity of this clone for the homologous host. Only the Syrian golden hamster (Mesocricetus auratus) could be infected experimentally, though the mean intensity of infection was lower. Other heterologous recipients, mice and rats of one outbred and one inbred strain each, could not be infected. Even immunodeficient mice (athymic and C.B-17-scid) remained uninfected after inoculation of 5 x 10(5) cysts per mouse. This is the second Spironucleus clone, after the rat isolate (Schagemann et al., 1990), with a high level of host specificity suggesting heterogeneity within the genus Spironucleus.

Induction of the 68 kDa major heat-shock protein in different Theileria annulata- and virus-transformed bovine lymphoblastoid cell lines.

Expression of the major inducible heat-shock protein of 68 kDa (hsp68) has been analyzed in peripheral blood mononuclear cells (PBMC) from cattle and in six Theileria annulata- and two bovine leukemia virus-transformed bovine lymphoblastoid cell lines (BoLCL). By metabolic labeling, hsp68 could be detected in PBMC and BoLCL only after heat-shock, but not under normal culture conditions. Immunoblot analysis with an hsp68 reactive monoclonal antibody similarly revealed a strong hsp68 response after heat-shock in BoLCL, and no hsp68 expression under normal culture conditions. Normally kept PBMC, however, were weakly positive with the antibody. The data are discussed with respect to the constitutive expression of hsp68 seen in several other cell lines.

Demonstration of extrachromosomal DNA from Babesia equi merozoites.

An extrachromosomal nucleic acid element was detected in high-molecular-weight DNA preparations form Babesia equi merozoites. This extrachromosomal element was shown to be DNA rather than RNA and had an apparent fragment size of about 9 kilobase-pairs (kb). Hybridization experiments using purified 9-kb DNA as a probe revealed sequence homologies with extrachromosomal DNA from two other Babesia species.

Host specificity of Giardia muris isolates from mouse and golden hamster.

One isolate of Giardia muris from a naturally infected laboratory mouse (Mus musculus) and one from a naturally infected golden hamster (Mesocricetus auratus) were passaged three times by the inoculation of ten cysts (the minimal infectious dose) into barrier-maintained homologous hosts. Both of the resultant isolates were tested for infectivity by intragastric inoculation of 3-5 x 10(5) cysts into 40 mice (2 inbred strains), 40 rats (2 inbred strains), and 19 golden hamsters (1 outbred strain). Rats were not susceptible to infection with either isolate. Mice and golden hamsters did develop infections following their inoculation with the heterologous isolates. The mean intensity of heterologous infections with the hamster isolates was significantly lower than that of homologous infections. The mouse isolate induced a higher mean intensity of infection in hamsters as compared with homologous recipients. The mean intensity of infections induced by both isolates was greater in male hamsters than in females.

Enrichment of Babesia caballi-infected erythrocytes from microaerophilous stationary-phase cultures using Percoll gradients.

A rapid and simple method for concentrating leucocyte-free Babesia caballi-infected erythrocytes from in vitro cultures is described. Infected erythrocytes amounted to at least 95% of all red cells obtained.

Experimental infections in chickens with Chilomastix gallinarum, Tetratrichomonas gallinarum, and Tritrichomonas eberthi.

Flagellates from the caeca of a diseased hen and a diseased goose were transmitted to 35 specific pathogen-free (SPF) chickens. The flagellates of chicken origin were identified as Chilomastix gallinarum, Tritrichomonas eberthi, and Tetratrichomonas gallinarum. T. eberthi was not detected in the material of goose origin. Morphologic studies did not reveal any differences between Chilomastix and Tetratrichomonas specimens from chicken or goose origin. The species from the goose were identified as C. gallinarum and T. gallinarum (Syn. T. anseris Hegner, 1929). Both trichomonad species produced pseudocysts that developed in the faeces of chickens within 3 h after excretion. Only 17% of the trichomonads excreted had reached the pseudocyst stage. All three flagellate species are infective to chickens when inoculated per rectum or per os or when consumed with chlorinated tap water. The prepatency period was always less than 24 h. SPF chickens between 2 and 30 days of age were equally susceptible. The infections persisted at a high level of intensity throughout the observation periods, i.e. up to 7 months. Of 35 inoculated SPF chickens, 2 developed disease (emaciation, ruffled feathers, diarrhoea, dilatation of the caeca). The three flagellate species were cultivated in Diamond's medium for 110 days. Cryopreserved and cultivated flagellates retained their infectivity to chickens.

Haemoparasites of equines: impact on international trade of horses.

The geographical distribution of Babesia equi and Babesia caballi and their tick vectors is discussed. Control of infections with these protozoa is hampered by the lack of a suitable antiprotozoal drug and a reliable serological test. No vaccine is available. Ehrlichia risticii (the causal agent of Potomac horse fever) and E. equi are rickettsial parasites which are difficult to control. Little is known of their geographical distribution and vectors. Early diagnosis is required for tetracycline therapy to be effective and there is a need for a rapid test to provide an early diagnosis.

Host specificity of cloned Spironucleus muris in laboratory rodents.

With three clones of Spironucleus muris (S. muris)--established from a mouse, hamster, and rat--homologous and heterologous host species were experimentally infected. Each host was susceptible to the clone originating from the homologous donor. In addition, both mice and hamsters were susceptible to the reciprocal heterologous clones. In contrast, infections of the rat with both heterologous clones were very poor, i.e. quantitatively low and ephemeral. It was not possible to infect hamsters and mice, not even athymic, with S. muris from the rat. This suggests a strain heterogeneity within the genus S. muris. In general, the genetic background of the host influenced the infection, the sex of the host did not.

Interaction between parasite and tick vector.

Interaction of tick vectors with Borrelia species including B. burgdorferi, rickettsias and piroplasms has been demonstrated by describing selected phenomena. In particular, the various environments inside the tick vector have been considered, including the midgut lumen, gut epithelial cells, body cavities and tissues. Intracellular parasitism occurs in different compartments of the host cell: parasitophorous vacuoles (Anaplasma marginale), phagolysosomes (Coxiella spp.), cytoplasm (Rickettsia, spp. piroplasms) or nucleus (some rickettsiae).

Quantitative description of the development of Babesia ovis in Rhipicephalus bursa (hemolymph, ovary, eggs).

The development and infection dynamics of Babesia ovis in the hemolymph, ovaries, and eggs of Rhipicephalus bursa are described quantitatively, based mainly on examination of Giemsa-stained smears. After alimentary infection of female ticks, their hemolymph became infected 5 days after repletion (p.repl.). The prevalence and mean intensity of infection increased during the course of infection studied, up to 17 days p.repl. After vertical infection of female ticks, their hemolymph was infected only during the first 3 days after the onset of infestation (p. infest.) and again after the onset of alimentary infection 5 days p.repl. There was a positive correlation between prevalence and mean intensity of infection in the hemolymph. The prevalence of infection decreased with aging of the unfed adult ticks. After alimentary infection, the ovaries became infected 6 days p.repl., and after vertical infection, 3 days p. infest; they remained infected until the death of the tick. Ticks selected for susceptibility during 18 and 19 vertically infected generations were more susceptible than ticks in their first to third vertically infected generations or alimentarily infected ticks. Eggs deposited on day 1 of oviposition were noninfected after alimentary infection of the female tick. After vertical infection of the tick, even such eggs became infected; the infection, then, was detectable in eggs produced throughout the oviposition period regardless of the infection mode. Intense hemolymph infections induced an increase of egg degeneration and a decrease of total as well as infected egg production. There was a positive correlation between the number of deposited and infected eggs as well as between prevalence and mean intensity of infection in eggs.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmission of Megatrypanum trypanosomes to Cervus dama by Tabanidae.

Four fallow deer, Cervus dama, became infected with Trypanosoma (megatrypanum) sp. by oral application of triturated guts from tabanids collected in an area with deer but without any cattle; four control calves remained negative. Upon challenge with triturated guts from tabanids from an area with pastured cattle, the four calves became infected with Trypanosoma (M.) theileri. The prepatent period in deer was five days or less. Haematopota spp. and Tabanus spp. were identified as vectors of the deer trypanosomes. It is concluded that the trypanosomes of C. dama belong to a Megatrypanum species that is not identical with T. theileri.