Comparative ultrastructural studies onBesnoitia besnoiti andBesnoitia caprae

Comparative transmission electron microscopy onBesnoitia besnoiti and on a strain ofBesnoitia derived from goats in Kenya revealed that the two organisms differ in their pellicle, micropore, microtubules, nucleus, wall-forming body 1 (W1), amount of lipids and amylopectin. Thus the caprine besnoitia is probably a different organism and the termBesnoitia caprae should continue to be used.     

Caprine besnoitiosis: an emerging threat and its relationship to some other infections of ungulates by Besnoitia species

Caprine besnoitiosis, caused by the cyst-forming protozoal apicomplexan Besnoitia caprae appears to be endemic in Kenya, Nigeria and Iran, but has yet to be detected in other parts of the world. The infection causes an important parasitic disease of goats in affected developing countries. Bovine besnoitiosis, is a widespread disease of cattle in Africa, Asia (but not Iran) and southern Europe. Recent epidemiological data confirm that the incidence and geographical range of bovine besnoitiosis in Europe is increasing, which is why growing attention has been given to the condition during the past decade. This paper reviews pertinent information on the biology, epidemiology, pathology, clinical signs, diagnosis and control of caprine besnoitiosis, together with its similarities to, and differences from, bovine besnoitiosis. The serious economic consequences of besnoitiosis on goat breeding and local meat and hide industries is also considered.     

Bovine besnoitiosis in Germany

This paper reports a case of natural occurring bovine besnoitiosis in Germany. The skin lesions consisted of multifocal hypotrichosis and alopecia, lichenification, erythema and seborrhoea. Histopathologic findings revealed characteristic cysts of Besnoitia spp. The diagnosis was confirmed by serology and the species Besnoitia besnoiti was identified by polymerase chain reaction (PCR).     

Experimental transmission of Besnoitia caprae in goats

Experimental transmission of Besnoitia caprae from naturally chronically-infected goats to susceptible ones was achieved by intra-nasal instillation and intra-conjunctival inoculation of cystozoite-containing suspensions, subcutaneous implantation of fascia containing cysts and alternate needle pricking between the infected and non-infected goats. Typical chronic symptoms developed in the fascia-infected does. Cystozoite inoculation into the eyes and mouth did not result in infection. Kids born of dams with acute and chronic besnoitiosis did not contract the infection in utero, suggesting that intra-uterine transmission may not occur. In contrast to does with acute besnoitiosis, which occasionally aborted, the does with chronic besnoitiosis gave birth to healthy kids. Kids below the age of 4 months (pre-weaned period) born of both infected and non-infected does were susceptible to besnoitiosis but appeared to be more resistant than adult goats.     

Enterotoxaemia in goats

Enterotoxaemia of sheep and goats occurs worldwide, but the condition in goats is poorly understood. The disease in goats is mostly caused by Clostridium perfringens type D, although the role of the toxins of this microorganism in the pathogenesis of the disease is not fully understood. The disease occurs in three forms, peracute, acute and chronic, the cardinal clinical sign of the acute and chronic forms being diarrhoea. The main biochemical alterations are hyperglycaemia and glycosuria, while at necropsy the disease is often characterized by haemorrhagic colitis. The typical histological changes observed in the brain of sheep with enterotoxaemia are not considered to be a common feature of enterotoxaemia in goats. Although the pathogenesis of caprine enterotoxaemia has not yet been properly defined, it is usually accepted that the presence of C. perfringens type D in the small bowel, together with a sudden change to a diet rich in carbohydrates, is the main predisposing factor for the disease. Vaccination seems to be poorly effective in preventing caprine enterotoxaemia, which might be due to the fact that the enteric form of the disease is partially independent of circulating C. perfringens toxin. More studies are needed on caprine enterotoxaemia, especially of its pathogenesis and immunity, in order to develop more efficient control measures for this disease.     

Quantitative real time polymerase chain reaction assays for the sensitive detection of Besnoitia besnoiti infection in cattle

Bovine besnoitiosis, an economically important disease in cattle in some countries of Africa and Asia, is emerging in Europe. The definitive host of Besnoitia besnoiti, the causative agent of bovine besnoitiosis, is unknown and the transmission of the parasite is not completely understood. Sensitive and quantitative DNA detection methods are needed to determine whether serologically positive animals are infectious and to examine the role of vectors (e.g. haematophagous insects) in the transmission of the parasite. To this end, we established two different 5'-nuclease quantitative assays to detect B. besnoiti infection in cattle and to estimate the parasite load in samples (BbRT1 and BbRT2). These PCRs are based on the sequence of the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene. Tests with serial dilutions of B. besnoiti genomic DNA in a buffer containing 100 ng/μl bovine DNA revealed a detection limit of 0.01 pg genomic B. besnoiti DNA. Reliable quantification was possible in samples containing ≥1 pg B. besnoiti genomic DNA with a coefficient of variation of ≤ 2%. To estimate the diagnostic sensitivity of the tests, skin biopsies and scrapings from the mucous membrane of the vestibulum vaginae (vaginal scrapings) were taken from cattle with clinical signs of chronic besnoitiosis. Regardless of the real time PCR assay used, 90.7% (39/43) of these animals were positive in at least one of two samples (skin or vaginal scrapings). Antibody titers, as determined by an immunofluorescent antibody test, and the threshold cycle values of the real time PCR obtained for skin samples and vaginal scrapings, were significantly correlated. The specificity of the PCRs was confirmed using genomic DNA from related parasites, including genomic DNA of Besnoitia spp., Neospora caninum, Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Isospora spp., Sarcocystis spp., Eimeria bovis, Cryptosporidium parvum, and Trypanosoma brucei brucei. Since the sequence of the ITS-1 region of B. besnoiti is identical with that of Besnoitia species isolated from donkeys (Besnoitia bennetti), and reindeer (Besnoitia tarandi), both real time PCRs detected also DNA of these parasites. One of the B. besnoiti real time PCRs, BbRT1, but not BbRT2, cross-reacted with Besnoitia darlingi, Besnoitia oryctofelisi, and Besnoitia neotomofelis when large amounts of genomic DNA (10 ng) were used. The other B. besnoiti real time PCR assay (BbRT2) was specific for B. besnoiti, B. bennetti and B. tarandi, but did not react when 10 ng DNA of other related parasite species from the genus Besnoitia or other genera were subjected to analysis.     

Besnoitiosis of the reproductive tract of a Blue Duiker (Cephalophus monticola)

A mature male Blue Duiker that had been born in the United States was submitted for necropsy examination following a brief illness. On histologic examination of the reproductive tract several Besnoitia cysts were found in the epididymis, prostate and bulbourethral gland. The lack of an inflammatory response or any negative effect on fertility, based on histologic evaluation and breeding history, is in contrast with the severe orchitis, epididymitis and infertility of besnoitiosis in cattle. This is the first report of an autochthonous infection of Besnoitia in the United States as well as the first report of besnoitiosis in a Blue Duiker.     

Standardization of the outbred BALB/c mice as a suitable animal model for <Emphasis Type="BoldItalic">Besnoitia caprae</Emphasis> studies

The Apicomplexan parasite Besnoitia caprae infects wild and domestic goats. Knowledge on Besnoitia caprae specially an optimized animal model is sparse. Experimental infections with tachyzoites of BC-Pars obtained from BALB/c mice were conducted in outbred mice to determine the infectivity and LD50 of Besnoitia caprae. Six groups of five mice were intraperitoneally infected with 12.5 × 10³, 25 × 10³, 5 × 10⁴, 1 × 10⁵ and 2 × 10⁵ tachyzoites and a control inoculum of DMEM, respectively. Although morbidity and mortality were observed in all groups, two mice in the 12.5 × 10³ group showed alopecia and skin lesions on 60 days post-infection (DPI). Histopathological and molecular examination of skins confirmed B. caprae infection. The LD₅₀ was calculated as 25 × 10^3.2 tachyzoites per mouse. The results indicate that outbred BALB/c mice can be used as a suitable model of besnoitiosis and to screen candidate treatments and to test the efficacy of vaccines for besnoitiosis.     

The Early Effects of Clostridium perfringens Type D Epsilon Toxin in Ligated Intestinal Loops of Goats and Sheep

Clostridium perfringens type D produces enterotoxaemia in goats, sheep and other animals. The disease is caused by C. perfringens epsilon toxin and, while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by the ovine than by the caprine intestine. In an attempt to clarify this, we examined the early effects of epsilon toxin on caprine and ovine intestine. Intestinal loop assays were performed to analyse the physiological and morphological changes induced by epsilon toxin in the intestine of these species. Fluid accumulation was observed in caprine and ovine ileum and colon treated with epsilon toxin. Ileal loops from goats treated with epsilon toxin retained sodium and water earlier than ovine ileal loops treated with the same toxin. Histological analysis showed morphological alterations in the colon of both species as early as 2 h after the commencement of epsilon toxin treatment; these changes were more marked in goats than in sheep. No morphological changes were observed in the ileum of either species after 4 h incubation with epsilon toxin. These results suggest that epsilon toxin modifies ion and water transport in the small and the large intestine of goats and sheep through different mechanisms.     

Evaluation of a commercial ELISA for the specific detection of antibodies against Besnoitia besnoiti

Bovine besnoitiosis is an economically important disease in cattle caused by the protozoan parasite Besnoitia besnoiti, which occurs endemically in many countries of Africa and Asia and is spreading in Europe. Serological identification of subclinically infected cattle is important to avoid the introduction of infected animals into naive herds. Here we determine the sensitivity and specificity of the PrioCHECK^® Besnoitia Ab, a serological test recently introduced into the European market. Analytical specificity was examined using sera from animals experimentally infected with parasites related to B. besnoiti (n = 27). Three animals experimentally infected with Neospora caninum or Toxoplasma gondii showed inconclusive reactions in the ELISA (percent positivity relative to the positive control [PP] 10% ≤ 20%) while all other sera reacted negative (PP < 10%). An estimate of the diagnostic specificity was obtained by analysing field sera from bovine herds without besnoitiosis but with abortion problems associated to N. caninum (n = 403). The analysis revealed a specificity of 94.3% or 96.8% depending on the applied cut-off (PP 10% or 20%, respectively). Sensitivity was assessed with sera from 110 animals of a herd in Germany where clinical bovine besnoitiosis was first diagnosed in September 2008. A positive serological reference standard was defined regarding sera from animals as reference positive, if these animals had tested positive in at least two of a panel of three other serological tests (two different B. besnoiti immunoblots and one immunofluorescence antibody test) on both of two sampling dates, November 2008 and April 2009. A diagnostic sensitivity of 91.8% or 75.5% was determined for sera collected in November 2008 and a sensitivity of 82.7% or 50% for sera collected in April 2009 (cut-off PP 10% or PP 20%, respectively). The marked drop in sensitivity from November 2008 to April 2009 was predominantly observed in reference-positive cattle without clinical signs. We conclude that PrioCHECK^® Besnoitia Ab is a valuable diagnostic tool to detect clinically infected animals. Thus it may be used to support control measures, e.g., for the separation of infected animals from the remaining herd to avoid a further transmission of the infection within the herd.     

Serodiagnosis of bovine besnoitiosis by ELISA and immunofluorescence tests

Sera from non-infected cattle and cattle infected with Anaplasma, Babesia, Theileria and Sarcocystis were tested for antibodies to Besnoitia in ELISA and immunofluorescence tests (IFT) with Besnoitia besnoiti of blue wildebeest origin as antigen. Only 2 out of 86 sera gave false positive reactions in ELISA and none in the IFT, indicating a high specificity for the tests. Three-hundred-and-three bovine sera from 3 farms in an area endemic for besnoitiosis were similarly tested and the results were correlated with clinical findings based on visual inspection for typical symptoms and the presence of cysts in the scleral conjunctiva. Most of the positive tests were observed in cattle older than 1 year. Of the cases with scleral cysts, 68,7% were positive in the ELISA and 81,74% in the IFT. However, 45,74% (ELISA) and 49,47% (IFT) of the clinically negative cattle were clinically positive, indicating a high incidence of clinically inapparent infection. These results indicate a relatively low sensitivity for these serological tests. An unexpected finding was that the ELISA remained negative for at least 60 days after experimental infection of the cattle, the maximum period for which tests were done, whereas the IFT became positive. No antibodies against B. besnoiti could be found in human sera. Besnoitia jellisoni antigen gave positive results with B. besnoiti antibodies in ELISA, but not in the IFT.     

Evidence for bovine besnoitiosis being endemic in Italy--first in vitro isolation of Besnoitia besnoiti from cattle born in Italy

Until 2009, bovine besnoitiosis had never been considered endemic in Italy and the only report on the disease in this country referred to animals imported from France shortly before. However, recently, an autochthonous outbreak of bovine besnoitiosis was reported in four herds located at the intersection of the borders between Emilia-Romagna, Toscana and Marche (Northern Apennine Mountains), which has led to an increased awareness concerning this disease. The present study describes a further outbreak of bovine besnoitiosis in Italy. The afflicted herd was a dairy herd with no evidence for contact with cattle from regions known to be endemic for bovine besnoitiosis. The farm investigation was initiated after a three-year old Holstein Friesian dairy cow with generalized thickening and lichenification of the skin was diagnosed with bovine besnoitiosis. The clinical diagnosis was confirmed by gross pathology, histopathology, serology and PCR. Bradyzoites released from tissue cysts obtained from the skin of this animal enabled the first in vitro isolation of Besnoitia besnoiti in Italy. This isolate was named Bb-Italy1. Sequencing of a 2118 bp spanning region including the complete internal transcribed spacer 1 and parts of the 18S and the 5.8S rRNA gene from DNA extracted from skin-derived zoites revealed a 99.9% identity to sequences known for other B. besnoiti isolated from cattle in Europe. Two GKO mice which had been inoculated intraperitoneally with bovine skin-derived bradyzoites became ill 7 days post inoculation. Parasitophorous vacuoles with multiplying zoites were observed in the cell culture inoculated with peritoneal fluids of these mice and a B. besnoiti infection in the mice and in the cell culture could be confirmed by real-time PCR. A serological investigation in the afflicted herd using immunoblots and an immunofluorescent antibody test (IFAT) revealed an overall herd seroprevalence of 9.7% (31/321), whereas within the female animals older than 2 years 17.0% (29/171) of the dams were tested positive. With one exception, an imported cow from Germany, all the seropositive animals were born in Italy. In connection with previously described autochthonous cases of bovine besnoitiosis the case described herein suggests that bovine besnoitiosis should be considered endemic in Italy.     

State-of-the-art review of goat TSE in the European Union, with special emphasis on PRNP genetics and epidemiology

Scrapie is a fatal, neurodegenerative disease of sheep and goats. It is also the earliest known member in the family of diseases classified as transmissible spongiform encephalopathies (TSE) or prion diseases, which includes Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE), and chronic wasting disease in cervids. The recent revelation of naturally occurring BSE in a goat has brought the issue of TSE in goats to the attention of the public. In contrast to scrapie, BSE presents a proven risk to humans. The risk of goat BSE, however, is difficult to evaluate, as our knowledge of TSE in goats is limited. Natural caprine scrapie has been discovered throughout Europe, with reported cases generally being greatest in countries with the highest goat populations. As with sheep scrapie, susceptibility and incubation period duration of goat scrapie are most likely controlled by the prion protein (PrP) gene (PRNP). Like the PRNP of sheep, the caprine PRNP shows significantly greater variability than that of cattle and humans. Although PRNP variability in goats differs from that observed in sheep, the two species share several identical alleles. Moreover, while the ARR allele associated with enhancing resistance in sheep is not present in the goat PRNP, there is evidence for the existence of other PrP variants related to resistance. This review presents the current knowledge of the epidemiology of caprine scrapie within the major European goat populations, and compiles the current data on genetic variability of PRNP.     

First in vitro isolation of Besnoitia besnoiti from chronically infected cattle in Germany

Besnoitia besnoiti was in vitro isolated during the first recorded outbreak of bovine besnoitiosis in Germany. Molecular characterization of the new isolate, named Bb-GER1, revealed almost 100% identity with other B. besnoiti isolates obtained in Portugal, Spain, Israel or South Africa, when partial sequences of the 18S ribosomal RNA gene, of the internal transcribed spacer 1 and of the 5.8S RNA gene were compared. Cystozoites obtained from skin tissue of one bull were infectious for γ-interferon knockout (GKO) mice by intraperitoneal (ip) inoculation. Tachyzoites were detected in the peritoneal cavity, spleen, liver and lung of the mice 5 days post-infection. The parasite could be maintained in GKO mice by ip inoculation for at least 5 passages. Peritoneal washings containing tachyzoites were obtained from infected mice and used to infect five cell lines (Vero, MARC-145, NA42/13, BHK₂₁, KH-R). The best growth of tachyzoites was observed in BHK₂₁ cells, but replication occurred to a smaller extent also in MARC-145, NA42/13 and KH-R cells. Subsequent comparative analyses revealed that after direct infection of these cell lines with cystozoites derived from bovine skin, the growth was best in NA42/13 cells. Considerable replication was also observed in the BHK₂₁ and KH-R cell lines. Our observations on the growth characteristics of Bb-GER1 partially contrast those for other isolates. The preferential growth in particular cell lines may be characteristic for particular B. besnoiti isolates. A potential association between growth properties and differences in virulence remains to be established. This is the first in vitro isolation of B. besnoiti from cattle in Germany.     

Serological comparisons of antigenically related herpesviruses in cattle,red deer and goats

Serological comparisons were made using related herpesviruses from cattle (bovid herpesvirus 1), red deer (herpesvirus of cervidae 1) and goats (bovid herpesvirus 6) by virus neutralization and enzyme-linked immunosorbent assays. The test samples comprised field sera from British cattle, red deer and goats and sera from experimentally infected or immunized animals. Both the cervine and caprine viruses appeared to be more closely related to bovid herpesvirus 1 than they were to each other. Cattle sera reacted most strongly with the bovine virus and deer sera with the cervine virus. Antibodies to the caprine virus were not detected in the samples from British goats.     

A longitudinal study of Besnoitia besnoiti infections and seasonal abundance of Stomoxys calcitrans in a dairy cattle farm of southwest France

Bovine besnoitiosis, caused by the cyst-forming apicomplexan Besnoitia besnoiti, is commonly reported in some restricted regions of South-Western Europe, and in larger regions of Africa and Asia. This infection is thought to be transmitted by blood feeding insects and is responsible for major economic losses in cattle production. A recent emergence in Europe, notified in the Centre of France, Spain and Germany, has attracted more attention to this disease. Clinical signs could appear in some animals; however, many infected cattle remain asymptomatic or show scleral-conjunctival cysts (SCC) only. Recent development of serological methods allows carrying out seroepidemiological field studies. In this respect, a long-term investigation was performed in a dairy cattle farm localized in an enzootic area of besnoitiosis of South-western France between March 2008 and May 2009. The objective was to estimate the seasonal pattern of B. besnoiti infections based on the presence of SCC and serology (ELISA and Western blot). In parallel, an entomological survey was conducted to describe population dynamics of Stomoxys calcitrans and Tabanidae species. The seroprevalence determined by Western blot in a cohort of 57 animals continuously present during the whole survey increased from 30% in March 2008 to 89.5% in May 2009 and was always higher than the prevalence based on clinically assessed SCC. New positive B. besnoitia seroconversions occurred throughout the year with the highest number in spring. In addition, many seroconversions were reported in the two months before turn-out and could be associated with a high indoors activity of S. calcitrans during this period.     

Identification and partial purification of soluble antigens from culture-grown Besnoitia besnoiti endozoites

Soluble antigens from culture-grown Besnoitia besnoiti endozoites were identified following their partial purification by affinity chromatography. A specific eluate obtained after affinity chromatography on a column to which antibodies from serum of a naturally infected cow were bound exhibited seven polypeptide bands on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Five bands were observed in the eluate from an immunoadsorbent to which antibodies from an experimentally infected calf were coupled. Eluted antigens were reactive in the enzyme-linked immunosorbent assay (ELISA). Reactivity of electrophoretically resolved antigens with sera of endozoite-inoculated cattle and sera from field cases of besnoitiosis were studied using the Western immunoblotting technique.     

Risk of exposure to Coxiella burnetii from ruminant livestock exhibited at Iowa agricultural fairs

Coxiella burnetii is a zoonotic pathogen typically associated with clinical and asymptomatic infection in ruminant livestock. A re‐emerging pathogen of significant public health importance, C. burnetii has caused recent epidemics in the United States and Europe, and public livestock exhibitions are increasingly scrutinized as a potential source of C. burnetii exposure. Although C. burnetii prevalence data among North American domestic ruminants are extremely limited, contemporary studies suggest that this pathogen is both geographically widespread and highly prevalent on a herd basis, especially in dairy cattle and goat populations. We utilized a real‐time PCR assay to detect C. burnetii faecal shedding by clinically normal, non‐periparturient beef cattle, meat goats and sheep exhibited at Iowa agricultural fairs. Individual faecal samples were collected from beef cattle, meat goats and sheep exhibited at twelve Iowa county fairs during the summer of 2009. The sample pool was blocked by species and fair, and ten samples from each block were randomly selected for the diagnostic assay; this test pool is considered sufficient to identify with 95% confidence a shedding animal in a population prevalence of 2.85% (cattle and sheep) and 6.25% (goats). Detection of C. burnetii DNA was determined through use of a real‐time PCR assay validated for use in bovine, ovine and caprine faeces; threshold of detection is one DNA copy per PCR (sensitivity 95.8%, specificity 100%). All tested samples were negative for C. burnetii DNA. We conclude that non‐dairy, non‐periparturient ruminants exhibited at Iowa fairs are unlikely to shed C. burnetii in their faeces and that this population should not be considered to be a significant exposure risk to other livestock or fair attendees.     

Blood-feeding patterns of horse flies in the French Pyrenees

Horse flies can mechanically transmit Besnoitia besnoiti, the agent of bovine besnoitiosis. Although previously limited to enzootic areas, especially the French Pyrenees Mountains, bovine besnoitiosis is now considered a re-emerging disease in western Europe. To improve understanding of the role of horse flies as mechanical vectors, this study investigated their blood-feeding ecology in the eastern French Pyrenees, in two high-altitude summer pastures whose main domestic ungulates were cattle, and in a wildlife park with native fauna. Species-specific PCR assays were conducted to identify the sources of blood meals: wild boar, horse, cattle (or bison), sheep (or mouflon), goat, red deer, roe deer and izard (or Pyrenean chamois). In La Mouline pasture, tabanids (N = 20) fed on red deer (70%) and cattle (30%). In Mantet pasture, tabanids (N = 24) fed on cattle (52%), red deer (20%), wild boar (16%), horse (8%) and sheep (4%). In the wildlife park, Tabanus bromius (N = 32), the most abundant species collected, fed on red deer (85%), bison (9%) and wild boar (6%). Despite relatively high densities in both the pastures and in the wildlife park, small wild ungulates (izard, mouflon and roe deer) were not detected as a source of blood meals. Only two mixed blood meals were identified in two specimens of T. bromius: cattle/horse for the specimen collected in the pastures, and bison/wild boar for the specimen collected in the wildlife park. Our findings showed that tabanids display a level of opportunistic feeding behaviour, in addition to a preference for red deer, the latter being particularly true for Philipomyia aprica, the most abundant species collected in the pastures.