Prospective cohort study reveals unexpected aetiologies of livestock abortion in northern Tanzania.

Livestock abortion is an important cause of productivity losses worldwide and many infectious causes of abortion are zoonotic pathogens that impact on human health. Little is known about the relative importance of infectious causes of livestock abortion in Africa, including in subsistence farming communities that are critically dependent on livestock for food, income, and wellbeing. We conducted a prospective cohort study of livestock abortion, supported by cross-sectional serosurveillance, to determine aetiologies of livestock abortions in livestock in Tanzania. This approach generated several important findings including detection of a Rift Valley fever virus outbreak in cattle; high prevalence of C. burnetii infection in livestock; and the first report of Neospora caninum, Toxoplasma gondii, and pestiviruses associated with livestock abortion in Tanzania. Our approach provides a model for abortion surveillance in resource-limited settings. Our findings add substantially to current knowledge in sub-Saharan Africa, providing important evidence from which to prioritise disease interventions.

The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions.

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.

Genetic diversity and shedding profiles for Cryptosporidium parvum in adult cattle and their calves.

Cryptosporidiosis is an important disease in neonatal calves, causing watery diarrhoea, loss of appetite, and production losses. Dehydration from diarrhoea often results in the calf requiring rehydration or veterinary treatment to prevent calf mortality. Transmission of Cryptosporidium to calves still has some major knowledge gaps, such as the initial source of oocysts ingested by calves and how these oocysts can persist between calving periods. Some studies have examined the role of adult cattle in the transmission of Cryptosporidium oocysts, although these have yielded inconclusive results. In this study, highly sensitive oocyst extraction from faeces and detection techniques, sensitive to 5 oocysts per gram using a 50 g sample, were used to genotype faecal samples from adult cattle and their calves to determine if adult cattle could be a source of Cryptosporidium infection for their calves. On a dairy farm, faecal samples from adult cattle were collected twice per week for 0-3 weeks before calving and from their calves three times per week until they reached 3 weeks of age followed by twice per week until they reached 6 weeks of age. On a beef farm, samples were collected from both adults and calves at a single time point. Faecal samples were examined to compare species and multilocus genotypes of Cryptosporidium parvum. Results show that C. parvum was the most prevalent species on both the dairy and beef farms. The calves within each herd appear to have one predominant single multilocus genotype, whereas adult cattle have multiple distinct genotypes. Adult cattle on the dairy farm, tested before calving, in the majority of cases had a multilocus genotype that is different from that detected in their calves. On the beef farm, where samples were taken at the same time, the majority of adult cattle matched the multilocus genotype of their calves. This study shows that adult cattle display a higher diversity of C. parvum genotypes on both farms compared to the calves. The data also represent a detailed longitudinal prevalence study of the shedding profiles and genotype of Cryptosporidium parasites detected in dairy calves from birth to 6 weeks of age.

Early immune responses and parasite tissue distribution in mice experimentally infected with oocysts of either archetypal or non-archetypal genotypes of Toxoplasma gondii.

In most of the world Toxoplasma gondii is comprised of archetypal types (types I, II and III); however, South America displays several non-archetypal strains. This study used an experimental mouse model to characterize the immune response and parasite kinetics following infection with different parasite genotypes. An oral inoculation of 50 oocysts per mouse from T. gondii M4 type II (archetypal, avirulent), BrI or BrIII (non-archetypal, virulent and intermediate virulent, respectively) for groups (G)2, G3 and G4, respectively was used. The levels of mRNA expression of cytokines, immune compounds, cell surface markers and receptor adapters [interferon gamma (IFNγ), interleukin (IL)-12, CD8, CD4, CD25, CXCR3 and MyD88] were quantified by SYBR green reverse transcription-quantitative polymerase chain reaction. Lesions were characterized by histology and detection by immunohistochemistry established distribution of parasites. Infection in G2 mice was mild and characterized by an early MyD88-dependent pathway. In G3, there were high levels of expression of pro-inflammatory cytokines IFNγ and IL-12 in the mice showing severe clinical symptoms at 8­11 days post infection (dpi), combined with the upregulation of CD25, abundant tachyzoites and tissue lesions in livers, lungs and intestines. Significant longer expression of IFNγ and IL-12 genes, with other Th1-balanced immune responses, such as increased levels of CXCR3 and MyD88 in G4, resulted in survival of mice and chronic toxoplasmosis, with the occurrence of tissue cysts in brain and lungs, at 14 and 21 dpi. Different immune responses and kinetics of gene expression appear to be elicited by the different strains and non-archetypal parasites demonstrated higher virulence.

Detection of Toxoplasma gondii in retail meat samples in Scotland.

Toxoplasma gondii is a globally important zoonotic parasite ranked as one of the most significant causes of disease burden among the major foodborne pathogens. Consumption of undercooked meat is a well-known risk factor for infection so the aim of this study was to investigate the presence of T. gondii in meat samples from retail outlets in Scotland. In Sampling Period 1, 300 meat samples (39 beef, 21 chicken, 87 lamb, 71 pork and 82 venison) were purchased from butchers', farmers' markets, farm shops and supermarkets, and in Sampling Period 2, 67 pure venison samples only were purchased from farmers' markets, farm shops and supermarkets. DNA was extracted and screened for T. gondii using a quantitative PCR targeting the 529 bp repeat element, and any positive samples were genotyped using PCR-RFLP targeting 10 markers. Meat juice was screened for T. gondii antibodies using a commercial ELISA or modified agglutination assay. Toxoplasma gondii DNA was detected in 0/39 (0%) beef samples, 1/21 (4.8%) chicken samples, 6/87 (6.9%) lamb samples, 3/71 (4.2%) pork samples and 29/82 (35.4%; Sampling Period 1) and 19/67 (28.4%; Sampling Period 2) venison samples. Partial PCR-RFLP genotyping revealed both clonal and non-clonal genotypes. Antibodies to T. gondii were detected in the meat juice of 2/38 (5.3%) beef samples, 3/21 (14.3%) chicken samples, 14/85 (16.5%) lamb samples, 2/68 (2.9%) pork samples and 11/78 (14.1%; Sampling Period 1) and 8/50 (16%; Sampling Period 2) venison samples. This is the first study to report the presence of T. gondii in retail meat products in Scotland and has highlighted venison as a potentially high risk meat. Further work is required to determine viability of parasites in this particular meat product.

Long-term production effects of clinical cryptosporidiosis in neonatal calves.

Cryptosporidiosis can have a devastating effect in neonatal calves, resulting in diarrhoea, dehydration and, in severe cases, death of the animal. The disease is caused by Cryptosporidium spp. and is one of the most common causes of calf enteritis in the UK. The parasite is very difficult to remove from the farm, as the oocysts have a tough outer wall which enables the parasite to survive for several months in moist temperate environmental conditions and it is difficult to kill oocysts with common disinfectants used on a farm. If appropriate management practises are applied, the disease is usually self-limiting and most calves will recover. It has been shown, in studies with children and in lambs, that severe clinical cryptosporidiosis can result in long-term growth and cognitive impairment compared with individuals with no obvious signs of the disease. This study measured the long-term growth rate of beef calves on farm by comparing groups of animals that had suffered differing degrees of clinical severity of cryptosporidiosis as neonates. A group of 27 beef calves were enrolled in the study and monitored from birth to 6 months of age. The calves were scored for severity of cryptosporidiosis and weighed at regular intervals. The average difference in weight gain, at 6 months, between a group of calves that had severe cryptosporidiosis as neonates and a group of calves with no clinical signs of infection was 34 kg. Those calves that had experienced severe cryptosporidiosis as neonates showed a significantly reduced live weight gain compared with those calves showing no clinical signs of infection (P = 0.034). Therefore, the impact of severe cryptosporidiosis in neonatal calves has longer term effects on weight gain and production efficiency, resulting in the parasite having a greater impact on cattle production than previously thought.

A One Health Approach to Tackle Cryptosporidiosis.

Cryptosporidiosis is a significant diarrhoeal disease in both people and animals across the world and is caused by several species of the protozoan parasite Cryptosporidium. Recent research has highlighted the longer-term consequences of the disease for malnourished children, involving growth stunting and cognitive deficits, and significant growth and production losses for livestock. There are no vaccines currently available to prevent the disease and few treatment options in either humans or animals, which has been a significant limiting factor in disease control to date. A One Health approach to tackle zoonotic cryptosporidiosis looking at new advances in veterinary, public, and environmental health research may offer several advantages and new options to help control the disease.

Cryptosporidium Prevalence in Calves and Geese Co-Grazing on Four Livestock Farms Surrounding Two Reservoirs Supplying Public Water to Mainland Orkney, Scotland.

The parasite Cryptosporidium parvum represents a threat to livestock health and production, water quality and public health. Cattle are known to be significant reservoirs of C. parvum, but transmission routes are complex and recent studies have implicated the potential role of wildlife in parasite transmission to cattle and water sources. On the Orkney Isles, high densities of Greylag geese (Anser anser) cause widespread faecal contamination of cattle pastures, where cryptosporidiosis is known to be the main cause of neonatal calf diarrhoea and Cryptosporidium contamination frequently occurs in two reservoirs supplying Mainland Orkney's public water. This study aimed to determine the Cryptosporidium species and subtypes present in geese and calves co-grazing on four farms surrounding two reservoirs on Mainland Orkney. Results indicated a high level of C. parvum prevalence in calves, geese and water samples. gp60 analysis illustrated that higher genotypic diversity was present in the goose population compared with calves, but did not yield sequence results for any of the water samples. It can be concluded that the high levels of C. parvum evident in calves, geese and water samples tested represents a significant risk to water quality and public health.

Cell mediated and innate immune responses in pigs following vaccination and challenge with Toxoplasma parasites.

Toxoplasma gondii has a worldwide distribution and can infect almost all warm blooded animals including pigs and humans. This study aims to examine the immune responses induced in pigs following vaccination (live S48 tachyzoites) and/or challenge with T. gondii oocysts, through the examination of changes in levels of transcription in CD4, CD8α, IFN-γ, IL-12p35, CXCR3, MyD88. The experiment involved four groups of animals; pigs in group 1 (Challenged) (Chal) were challenged orally with (1 × 103 oocysts) on day 28 of the experiment. Pigs in group 2 (Vaccinated /Challenged) (Vac/Chal) were vaccinated (S48 isolate tachyzoites) on day 0, then challenged on day 28. The group 3 (Vaccinated) (Vac) animals were vaccinated (S48 isolate tachyzoites) on day 0 of the experiment. Finally the group 4 (control) pigs remained non-vaccinated and non-challenged. All animals were culled 6 weeks post challenge. At post mortem samples of retropharyngeal lymph node (RLN), mesenteric LN (MLN) and spleen were collected, RNA was extracted and cDNA synthesised. The results showed significant increases in IFN-γ expression in samples from groups 1 (Chal) and 2 (Vac/Chal) (RLN) and groups 1, 2 and 3 (Vac) (spleen) and in MyD88 expression (RLN) in samples from groups 1, 2 and 3 compared to the group 4 (control) animals. Significant increases were also observed in CD8α expression in group 1 (Chal) (RLN) and groups 1 and 2 (Vac/Chal) (RLN and MLN) compared against group 4 (control) and group 3 (Vac) respectively. Conversely, significant down regulation of CD4 and/or IL-12p35 transcription was found in at least one sample from groups 1 (Chal), 2 (Vac/Chal) and 3 (Vac) compared to group 4 (control) pigs. This study demonstrates that cell mediated and innate immune responses are generated in pigs following exposure to T. gondii parasites (oocysts or tachyzoites), key amongst them appear to be IFN-γ, MyD88 and CD8α.

Global selective sweep of a highly inbred genome of the cattle parasite Neospora caninum.

Neospora caninum, a cyst-forming apicomplexan parasite, is a leading cause of neuromuscular diseases in dogs as well as fetal abortion in cattle worldwide. The importance of the domestic and sylvatic life cycles of Neospora, and the role of vertical transmission in the expansion and transmission of infection in cattle, is not sufficiently understood. To elucidate the population genomics of Neospora, we genotyped 50 isolates collected worldwide from a wide range of hosts using 19 linked and unlinked genetic markers. Phylogenetic analysis and genetic distance indices resolved a single genotype of N. caninum Whole-genome sequencing of 7 isolates from 2 different continents identified high linkage disequilibrium, significant structural variation, but only limited polymorphism genome-wide, with only 5,766 biallelic single nucleotide polymorphisms (SNPs) total. Greater than half of these SNPs (∼3,000) clustered into 6 distinct haploblocks and each block possessed limited allelic diversity (with only 4 to 6 haplotypes resolved at each cluster). Importantly, the alleles at each haploblock had independently segregated across the strains sequenced, supporting a unisexual expansion model that is mosaic at 6 genomic blocks. Integrating seroprevalence data from African cattle, our data support a global selective sweep of a highly inbred livestock pathogen that originated within European dairy stock and expanded transcontinentally via unisexual mating and vertical transmission very recently, likely the result of human activities, including recurrent migration, domestication, and breed development of bovid and canid hosts within similar proximities.

Shedding of Cryptosporidium in calves and dams: evidence of re-infection and shedding of different gp60 subtypes.

One of the most common causes of calf diarrhoea is the parasite Cryptosporidium parvum. Two longitudinal studies were carried out on a dairy farm Scotland to determine the prevalence of Cryptosporidium species and subtypes in a group of calves and to determine whether dams were a possible source of calfhood infection. Fecal samples were collected from 25 calves from birth to 12 months in the first year. In the second year, fecal samples were collected from pregnant cows (n = 29) and their calves (n = 30) from birth to 6 months. The samples were tested for Cryptosporidium and speciated. Cryptosporidium parvum-positive samples were subtyped by GP60 fragment analysis. All calves in both studies shed Cryptosporidium during the study period. Cryptosporidium parvum was the predominant species detected in calves ⩽6 weeks of age and at 6 months of age, C. bovis and C. ryanae were detected in calves older than 4 weeks of age but ⩽6 months of age. The prevalence of Cryptosporidium was higher in younger animals than in older animals. GP60 subtyping revealed two subtypes in calves on this farm (IIaA15G2R1 and IIaA19G2R1) that differed in frequency by age. Adult cattle also shed C. parvum, of four gp60 genotypes.

Comparative virulence of Caribbean, Brazilian and European isolates of Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is a zoonotic parasite of global importance. The outcome of infection in humans can depend on a number of factors including the infecting stage of the parasite, inoculating dose and virulence of the infecting strain. Molecular epidemiological studies have demonstrated an abundance of atypical strains of T. gondii in South America, many of which have been associated with more severe sequelae of infection. The aim of this study was to compare the virulence of T. gondii strains isolated in the Caribbean to a virulent Brazilian strain and an avirulent European strain. METHODS: One hundred and twenty Swiss CD-1 mice were split into 8 groups of 15 mice and each group was inoculated with 200 tachyzoites of one of 8 isolates, comprising ToxoDB genotypes #1, #141, #265, #13, #3 and #6. Five mice per group were euthanized at day 8 post-inoculation (p.i.) and parasite burden was determined in heart, lungs and eyes using quantitative PCR. Lungs and brain were also examined by histopathology and immunohistochemistry. The remaining 10 mice per group were part of a survival experiment to assess virulence. DNA was extracted from tachyzoites of each of the 8 T. gondii isolates and genotyped at four ROP gene loci, including ROP5, ROP16, ROP17 and ROP18 to look for association with markers of virulence. RESULTS: Infection with ToxoDB genotype #13 from the Caribbean resulted in 100% of mice being euthanized which was comparative to infection with the virulent Brazilian strain (ToxoDB genotype #6). Significantly higher parasite burdens were recorded in the lungs and eyes of mice infected with ToxoDB genotypes #13 and #6. Genotyping of ROP loci revealed that the virulent Caribbean isolates had a different ROP18/ROP5 allelic profile (3/1) to the virulent Brazilian isolate (1/3); however, the avirulent Caribbean isolate (ToxoDB genotype #1) had the same ROP18/ROP5 profile as the avirulent European isolate (ToxoDB #3) (both 2/2). Caribbean isolates of intermediate virulence (ToxoDB #141 and #265) all had the same ROP18/ROP5 allelic profile (2/2). CONCLUSIONS: Isolates from the Caribbean with ToxoDB genotype #13 were acutely virulent for mice and comparable to a known virulent Brazilian isolate. The ROP protein allelic profile of the virulent Caribbean and Brazilian isolates differed indicating that perhaps other factors are involved in predicting virulence. Understanding virulence is important for predicting disease outcome in humans and may also aid vaccine design as well as drug discovery.

Detection of potentially human infectious assemblages of Giardia duodenalis in fecal samples from beef and dairy cattle in Scotland.

This study aimed to determine the prevalence and assemblages of Giardia duodenalis present in Scottish beef and dairy cattle at different ages, to try to ascertain if cattle could play a role in the spread of zoonotic assemblages of Giardia. A total of 388 fecal samples (128 beef and 253 dairy, seven of unknown breed) were collected from 19 farms in Scotland. Samples were sub-divided by host age, 1, 2, 3, 4, 5 and 6, 7-24 and ⩾25 weeks. DNA was extracted and tested by PCR to detect G. duodenalis DNA. Of the 388 samples, 126 tested positive, giving an overall prevalence of 32.5%, with positive samples being observed in all age groups tested. The prevalence in dairy cattle was 44.7% (113/235), which was significantly higher (P < 0.001) than the prevalence in beef cattle 10.1% (13/128). Sequence analysis demonstrated the presence of assemblage E (77.2%, sequence types E-S1-E-S5), assemblage B (18.2%) and assemblage A (sub-assemblages AI-AII) (4.6%). These data demonstrate that G. duodenalis is found routinely in both dairy and beef cattle throughout Scotland; the presence of assemblages A and B also indicates that cattle may play a role in the spread of potentially zoonotic assemblages of Giardia.

A one health approach to vaccines against Toxoplasma gondii.

Toxoplasmosis is a serious disease with global impact, now recognised as one of the most important food borne diseases worldwide and a major cause of production loss in livestock. A one health approach to develop a vaccination programme to tackle toxoplasmosis is an attractive and realistic prospect. Knowledge of disease epidemiology, parasite transmission routes and main risk groups has helped to target key host species and outcomes for a vaccine programme and these would be to prevent/reduce congenital disease in women and sheep; prevent/reduce T. gondii tissue cysts in food animal species and to prevent/reduce T. gondii oocyst shedding in cats. Most animals, including humans, develop good protective immunity following infection, involving cell mediated immune responses, which may explain why live vaccines are generally more effective to protect against T. gondii. Recent advances in our knowledge of parasite genetics and gene manipulation, strain variation, key antigenic epitopes, delivery systems and induction of immune responses are all contributing to the prospects of developing new vaccines which may be more widely applicable. A key area in progressing vaccine development is to devise standard vaccine efficacy models in relevant animal hosts and this is where a one health approach bringing together researchers across different disciplines can be of major benefit. The tools and technologies are in place to make a real impact in tackling toxoplasmosis using vaccination and it just requires a collective will to make it happen.

Detection and dissemination of Toxoplasma gondii in experimentally infected calves, a single test does not tell the whole story.

BACKGROUND: Although the detection of Toxoplasma gondii in bovine tissues is rare, beef might be an important source of human infection. The use of molecular techniques, such as magnetic capture qPCR (MC-qPCR), in combination with the gold standard method for isolating the parasite (mouse bioassay), may increase the sensitivity of T. gondii detection in infected cattle. The risk of transmission of the parasite to humans from undercooked/raw beef is not fully known and further knowledge about the predilection sites of T. gondii within cattle is needed. In the current study, six Holstein Friesian calves (Bos taurus) were experimentally infected with 106 T. gondii oocysts of the M4 strain and, following euthanasia (42 dpi), pooled tissues were tested for presence of the parasite by mouse bioassay and MC-qPCR. RESULTS: Toxoplasma gondii was detected by both MC-qPCR and mouse bioassay from distinct pools (100 g) of tissues comprising: liver, tongue, heart, diaphragm, semitendinosus (hindlimb), longissimus dorsi muscle (sirloin) and psoas major muscle (fillet). When a selection of individual tissues which had been used for mouse bioassay were examined by MC-qPCR, parasite DNA could only be detected from two animals, despite all calves showing seroconversion after infection. CONCLUSIONS: It is apparent that one individual test will not provide an answer as to whether a calf harbours T. gondii tissue cysts. Although the calves received a known number of infectious oocysts and highly sensitive methods for the detection of the parasite within bovine tissues were applied (mouse bioassay and MC-qPCR), the results confirm previous studies which report low presence of viable T. gondii in cattle and no clear predilection site within bovine tissues.

Molecular confirmation of Sarcocystis gigantea in a naturally infected sheep in Argentina: A case report.

The work describes a case of Sarcocystis gigantea infection in a 3-years-old Corriedale ewe from Buenos Aires Province, Argentina. The ewe was found dead with a poor body condition. Pathological and molecular studies were carried out in order to try and confirm the causative agent of the infection. At necropsy, approx. 100 whitish elliptic (3-5mm to 5-8mm) macrocysts with a hard consistency were observed along the esophageal and pharyngeal muscular layers. Microscopically, the macrocysts consisted of an eosinophilic wall, internal septa originated from the eosinophilic wall and basophilic parasitic cells were located among the septa. The sarcocysts were identified molecularly through PCR amplification and sequencing of a short segment of the 18S rRNA gene. Sequence analysis of the amplified DNA demonstrated 100% identity to S. gigantea sequences previously published. To our knowledge this is the first molecular confirmation of S. gigantea infection in sheep in the Americas.

The use of ELISA, nPCR and qPCR for diagnosis of ocular toxoplasmosis in experimentally infected pigs.

In the present study we experimentally infected pigs with T. gondii tachyzoites, bradyzoites and oocysts in order to evaluate IgG-ELISA, nested-PCR, and qPCR for diagnosis of ocular infection. Eighteen pigs were divided into four groups: G1 (infected with 103 tissue cysts of the M4 strain (type II) at day 28, n=5), G2 (infected with 103 oocysts of the M4 strain at day 28, n=5), G3 (infected with tachyzoites of S48 strain (type 1) at day 0, n=5), and G4 (uninfected unchallenged, control group n=3). At day 70 of the experiment all animals were culled, and serum, aqueous humor (AH) and vitreous humor (VH) samples were collected to perform indirect ELISA, and PCR (nPCR, and qPCR). By ELISA nine pigs (60%) out of 15 were positive in VH samples, and seven out of 15 (46%) were positive in AH samples. Both molecular techniques used here, nPCR and qPCR, were able to detect <50fg of T. gondii tachyzoite DNA. The nPCR and qPCR detected six (7/15, 47%) and two (2/15, 13.3%) positive animals respectively. Antibody responses were detected in serum and in AH and VH from the eye, suggesting that pigs may be an animal that could be used as a model to further our understanding of diagnosis of human ocular infection with T. gondii.

Bovine cryptosporidiosis: impact, host-parasite interaction and control strategies.

Gastrointestinal disease caused by the apicomplexan parasite Cryptosporidium parvum is one of the most important diseases of young ruminant livestock, particularly neonatal calves. Infected animals may suffer from profuse watery diarrhoea, dehydration and in severe cases death can occur. At present, effective therapeutic and preventative measures are not available and a better understanding of the host-pathogen interactions is required. Cryptosporidium parvum is also an important zoonotic pathogen causing severe disease in people, with young children being particularly vulnerable. Our knowledge of the immune responses induced by Cryptosporidium parasites in clinically relevant hosts is very limited. This review discusses the impact of bovine cryptosporidiosis and describes how a thorough understanding of the host-pathogen interactions may help to identify novel prevention and control strategies.

Detection of Babesia DNA in blood and spleen samples from Eurasian badgers (Meles meles) in Scotland.

Babesia are intraerythrocytic parasites of importance worldwide within the fields of human and veterinary medicine, as some Babesia sp., including Babesia microti are potentially zoonotic and can cause fatal disease in both humans and animals. The aims of this study were to use a nested PCR (amplifying the 18S rRNA gene) to determine the presence and species of Babesia parasite DNA found in blood (n = 47) and spleen (n = 47) samples collected from Eurasian badgers (Meles meles) in Scotland. The results showed 28/47 (59·6%) blood and 14/47 (29·8%) spleen samples tested positive for the presence of Babesia DNA. Initial sequence analysis of the Babesia DNA identified three distinct sequence types (submitted to GenBank KX528553, KX528554 and KX528555), which demonstrated ⩾99% identity to Babesia sp. parasites previously identified in badgers in Spain (KT223484 and KT223485). Phylogenetic analysis showed that the three isolates are closely related to Babesia annae, B. microti and other Piroplasmida species found in wildlife. Further sequence analysis of the samples demonstrated that the badgers were routinely infected with more than one parasite isolate and there was also evidence of genetic recombination between the Babesia parasite isolates (submitted to GenBank KY250472 - KY250477).

Predominance of atypical genotypes of Toxoplasma gondii in free-roaming chickens in St. Kitts, West Indies.

BACKGROUND: Toxoplasma gondii is a worldwide protozoan parasite of felids which can infect almost all warm-blooded animals, including humans. Free-roaming chickens are good indicators of environmental contamination with T. gondii oocysts because they feed from the ground. Previous research has demonstrated a high seroprevalence of T. gondii in domestic animals on St. Kitts but little is known about the genotypes circulating in the environment. METHODS: Hearts and brains from 81 free-roaming chickens in St. Kitts were digested and inoculated into 243 Swiss Webster mice in a bioassay. DNA was extracted from digested chicken tissues and the brains of all mice, and screened for T. gondii. Positive samples were genotyped using restriction fragment length polymorphism. Chicken sera were also screened for T. gondii antibodies using a modified agglutination test (MAT). RESULTS: Overall, 41% (33 out of 81) of chickens were positive for T. gondii either by serology and/or by PCR. Antibodies to T. gondii were detected by MAT in 32% (26 out of 81) of chickens, and T. gondii DNA was detected in mouse brains representing 26% (21 out of 81) of chickens. Genotyping of 21 DNA isolates, using polymorphisms at 10 loci, including SAG1, SAG2 (5'-3' SAG2 and alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico, revealed that 7 were ToxoDB genotype #141, 6 were #1 (Type II), 3 were #13, 3 were #265, one was #264 and one was #2 (Type III). Genotypes #13 and #141 appear to be more virulent. CONCLUSIONS: The results of this study highlight the greater genetic diversity of T. gondii circulating in the Caribbean region, with potentially different degrees of virulence to humans.