Cryptosporidium infections in suckler herd beef calves.

A study was carried out to investigate how common Cryptosporidium infections are in beef calves in Swedish suckler herds and to explore which species and subtypes that occur. We further aimed at identifying factors associated with shedding of Cryptosporidium oocysts in this type of calf management. The study was conducted in two regions in Sweden and included 30 herds. Faecal samples were collected from calves younger than 3 months. A brief clinical examination was done and a questionnaire was used to collect data on management routines. Faeces were cleaned and concentrated and oocysts identified by epifuorescence microscopy. Cryptosporidium positive samples were analyzed at the 18S rRNA and GP60 genes to determine species and Cryptosporidium parvum subtype, respectively. Logistic regression was used to identify factors associated with infection. Oocysts were detected in 122 (36.7%) calves from 29 (97%) herds, at 400 to 2.4 × 107 OPG. The youngest positive calves were only 1 and 2 days old. There was no association between age and Cryptosporidium infection. Cryptosporidium bovis, Cryptosporidium ryanae, C. parvum and Cryptosporidium ubiquitum were identified, with C. bovis being the major species. Two C. parvum subtypes, IIaA16G1R1 and IIdA27G1 were identified. Routines for cleaning calf pens and number of cows in calving pens were associated with infection.

Cryptosporidium parvum infections in a cohort of veterinary students in Sweden.

In March 2013, a veterinary student tested positive for Cryptosporidium; four classmates reported similar gastrointestinal symptoms. We aimed to identify source(s) and risk factors for Cryptosporidium infection in university persons symptomatic between 21 January and 14 April 2013. Sixty-four (79%) students from a cohort of 81 fourth-year veterinary students completed questionnaires, identifying 13 cases; four were Cryptosporidium parvum GP60 subtype IIaA16G1R1b, two were IIdA24G1, seven did not submit stool samples. Thirteen cases attended the university's field clinic before symptom onset (13/37 attendees, 35%); 11 visited at least one of four farms where students recalled seeing calves with diarrhoea. C. parvum subtype IIaA16G1R1b was identified in calves at one of the farms. Entering pens of calves with diarrhoea [relative risk (RR) 7·6, 95% confidence interval (CI) 1·7-33·5] and eating in clinic cars (RR 9·1, 95% CI 1·3-65·8) were associated with being a case. Washing hands at least twice per farm visit (0 cases, P = 0·03) was protective. This outbreak investigation was notable for rapid and effective collaboration between public health, veterinary and environmental sectors, leading to swift identification of a microbiological and epidemiological link between cases, infected calves and their farms. We recommend frequent hand-washing using proper technique and dissuasion from eating in clinic cars to minimize possible exposure to contaminated surfaces.

Is there a need for improved Cryptosporidium diagnostics in Swedish calves?

Cryptosporidium parvum is a common pathogen in preweaned calves but in Sweden Cryptosporidium bovis, which is considered apathogenic, is the most common species in this age group and it has been identified in diarrhoeal samples, indicating that it could be a cause of diarrhoea. In routine diagnostic procedures, infection is determined by microscopy, which is not sufficient to differentiate these species. We investigated whether routine Cryptosporidium diagnostic procedures need improvement to include species determination. The relation of Cryptosporidium spp. and subtype with the clinical picture and other pathogens was also investigated. A total of 782 diarrhoeal calf samples were analysed and Cryptosporidium infection was diagnosed in 198 samples. Cryptosporidium parvum was identified in 178, C. bovis in six and mixed C. bovis/C. parvum in seven samples. Twenty-seven C. parvum subtypes were identified, of which 16 were newly described. Except for three herds, only one subtype per herd was identified. Cryptosporidium parvum-positive calves were younger than C. bovis-positive calves and most C. parvum infections were seen at 1-3 weeks of age. Oocyst counts were higher in C. parvum samples. Yellow faecal colour was associated with C. parvum infection. Watery faeces had no greater association with C. parvum infection, but C. parvum subtype family IIa was more common than subtype family IId in watery faecal samples. No other pathogens were detected in the six C. bovis-infected calves, indicating a pathogenic potential. Our results show that species determination does not need to be included in routine Cryptosporidium diagnostic procedures in order to estimate the clinical relevance of infection in diarrhoeal calves. The maximum age when analysis for clinical cryptosporidiosis is performed can be lowered to 6 weeks of age. However, the indicated pathogenic potential of C. bovis warrants further attention.

Seroprevalence of Neospora caninum in free living and farmed red deer (Cervus elaphus) in Poland.

Serum samples from 47 free living and 106 farmed red deer (Cervus elaphus) from the Mazurian Lake District in north-east Poland were investigated for the presence of antibodies to Neospora caninum. A modified Neospora iscom-ELISA was used for initial screening. All sera with optical density (OD) values exceeding 0.400 absorbance units were further investigated by Western blot analysis. Eighteen sera were positive in both tests. Six of these were from free living and 12 from farmed animals giving prevalence of 13 and 11%, respectively. This is the first report of N. caninum infection in farmed and free-living red deer living in the same region where neosporosis was confirmed in cattle and the first evidence of exposure to the parasite in red deer in Poland.

Seroprevalence of Neospora caninum in gray wolves in Scandinavia.

Transmission of the protozoan parasite Neospora caninum between wild and domestic animals has gained some interest during recent years. Because of the close relationship between gray wolf (Canis lupus) and dog it has been suggested that gray wolf is a definitive host for the parasite. The aim of this study was to estimate the seroprevalence of N. caninum in Scandinavian gray wolves and to investigate any geographical patterns of the infection. The investigation was based on blood samples collected from 109 wolves between 1998 and 2009 within the Scandinavian wolf project Skandulv. They were analysed by N. caninum iscom ELISA and those with absorbance values exceeding 0.20 were also analysed by immunoblotting. Samples that were positive in both tests were deemed positive. Four (3.7%) wolves were positive at the first sampling. They were all sampled 2005 at different locations, and were both females and males. From one male wolf three samples were collected over a 7-year period. No antibodies were detected at the first sampling in 1998 when he was approximately 8 months old but when he was sampled again 5 and 7 years later the ELISA and immunoblotting were positive. The results indicate that N. caninum infection is present in Scandinavian wolves. It is unclear how the wolves acquired the infection.

The prevalence and spatial clustering of Neospora caninum in dairy herds in Norway.

The aims of this study were to estimate the national herd prevalence and the general spatial distribution of Neospora caninum infection in Norwegian dairy cattle. Bulk milk samples from 1657 randomly selected dairy herds were analysed by iscom ELISA for the presence of N. caninum specific antibodies. The national herd prevalence was estimated to be 0.7% (95% CI: 0.3-1.2%), which is the lowest national herd prevalence reported in Europe. In addition, we included a subpopulation study in which all 400 herds in the Oslo, Akershus and Østfold counties were sampled, and we found a herd prevalence of 1.8%. In the national herd study, a significant cluster of N. caninum infected herds was identified in Jaeren, on the south west coast of Norway. Jaeren has the highest density of farm animals in the country as well as climatic conditions that favour parasite survival. Dairy herds in this region had a 52 times higher relative risk of being N. caninum positive than dairy herds in other parts of Norway. This indicates that even though N. caninum infection is of minor importance in Norway, infection might cause abortion problems in high risk areas.

Endogenous and exogenous transplacental transmission of Neospora caninum - how the route of transmission impacts on epidemiology and control of disease.

Vertical transmission of the protozoan parasite, Neospora caninum is highly efficient and can take two forms - endogenous transplacental transmission resulting from activation of the quiescent bradyzoite stage during pregnancy or exogenous transplacental transmission resulting from ingestion of oocysts during pregnancy. Calves born carrying infection derived from either endogenous or exogenous transplacental transmission are capable of infecting their offspring when they start to breed. This review considers firstly the frequency with which exogenous and endogenous transmission occur, secondly the role of the immune response in controlling N. caninum infection and thirdly how the parasite persists in an immune-competent host and is re-activated during pregnancy.

Molecular typing of Cryptosporidium parvum associated with a diarrhoea outbreak identifies two sources of exposure.

An outbreak of cryptosporidiosis associated with exposure to outdoor swimming-pool water affected an estimated 800-1000 individuals. PCR products were obtained from faecal specimens from 30 individuals who tested positive for Cryptosporidium oocysts. RFLP and sequencing analyses showed that all individuals were infected with Cryptosporidium parvum. Among the infected individuals, five had just swum in an adjacent indoor pool during the same period, and had no identified contact with individuals linked to the outdoor pool. With the use of subgenotyping based on analysis of three mini- and microsatellite loci, MS1, TP14, and GP15, we could identify two sources of exposure. One subtype was associated with the outdoor pool and another with the indoor pool. These data demonstrate that the use of mini- and microsatellite loci as markers for molecular fingerprinting of C. parvum isolates are valuable in the epidemiological investigation of outbreaks.

Immunization of cattle with live tachyzoites of Neospora caninum confers protection against fetal death.

Neospora caninum is an obligate intracellular protozoan parasite that causes abortion in cattle. It is normally found as a latent infection controlled by a T-helper-cell type 1 response involving CD4(+) cytotoxic T cells and gamma interferon. Cattle may be infected by two different routes: transplacentally as a result of activation of the latent infection in the mother causing congenital infection or abortion and by ingestion of oocysts, which, if it occurs during gestation, can also result in abortion. Here, for the first time, we establish proof that live vaccination protects against fetal death, whereas immunization using whole-tachyzoite lysate in different adjuvants fails to protect against fetal death. Strong antibody responses were induced in all the vaccinated groups, and the quality and magnitude of these responses were similar in the live- and the lysate-vaccinated groups. In contrast, only the group immunized with live tachyzoites had strong cellular and gamma interferon responses prior to challenge, and these responses correlated with protection against fetopathy. These results suggest that a cellular immune response may be important in the mechanisms involved in protection against N. caninum-associated abortions.

Neospora caninum IgG avidity tests: an interlaboratory comparison.

Avidity tests can be used to discriminate between cattle that are acutely and chronically infected with the intracellular parasite Neospora caninum. The aim of this study was to compare the IgG avidity ELISA tests being used in four European laboratories. A coded panel of 200 bovine sera from well documented naturally and experimentally N. caninum infected animals were analysed at the participating laboratories by their respective assay systems and laboratory protocols. Comparing the numeric test results, the concordance correlation coefficients were between 0.479 and 0.776. The laboratories categorize the avidity results into the classes "low" and "high" which are considered indicative of recent and chronic infection, respectively. Three laboratories also use an "intermediate" class. When the categorized data were analysed by Kappa statistics there was moderate to substantial agreements between the laboratories. There was an overall better agreement for dichotomized results than when an intermediate class was also used. Taken together, this first ring test for N. caninum IgG avidity assays showed a moderate agreement between the assays used by the different laboratories to estimate the IgG avidity. Our experience suggests that avidity tests are sometimes less robust than conventional ELISAs. Therefore, it is essential that they are carefully standardised and their performance continuously evaluated.

A prospective study of the effect of Neospora caninum and BVDV infections on bovine abortions in a dairy herd in Arequipa, Peru.

We used a prospective seroepidemiological approach to investigate endemic abortion in a dairy herd in Arequipa, Peru, and its association with Neospora caninum and bovine viral-diarrhoea virus (BVDV) infections. Between January 2002 and March 2004, 1094 pregnancies were confirmed in 538 cows. Of these, 137 pregnancies (13%) in 121 cows ended in abortion. The serological status to N. caninum was assessed using a single serological screening, whereas BVDV status was assessed at the herd level through consecutive samplings of young stock. Cox proportional-hazards models were used to estimate the effect of N. caninum and BVDV on the hazard of early (between day 42 and day 100 in gestation), and late (after day 100) abortions, respectively. Serological status to N. caninum was included as a dichotomous variable, and the effect of BVDV estimated at the herd level, as a time-dependent seasonal effect. Because data from repeated pregnancies were included, we considered possible lack of independence between observations and included frailty effects into the models. Our models also considered the possible confounding by parity and animal origin. Only multiparity was associated with the hazard of early abortion (HR=2.8 compared to nulliparous heifers). N. caninum seropositivity significantly affected the hazard of late abortion, but interacted with parity. The HRs for Neospora-positive animals were 6.4, 3.7 and 1.9, respectively, for nulliparous heifers, first-lactation cows and multiparous cows. Evidence of BVDV circulating (or not) among the young stock was not associated with abortions, but few cows in this herd were susceptible to incident infection.

Supranational comparison of Neospora caninum seroprevalences in cattle in Germany, The Netherlands, Spain and Sweden.

Herd, within-herd and animal prevalences for Neospora caninum in beef and dairy cattle were compared between four countries. In randomly selected herds from regions of Germany, The Netherlands, Spain and Sweden that were representative for the cattle production of these countries, all animals > or = 2 years were examined serologically by enzyme-linked immunosorbent assays (ELISAs) with high test specificity (> 98.0%). In a previous study, the ELISAs had been validated against each other. Single reacting animals within a herd were confirmed by immunobloting. At the time of sampling, animal (age, breed, herdtype, sex, lactation stage) and herd data (region) were collected. Considerable differences in N. caninum herd, within-herd, and overall animal prevalence estimations were observed between countries, regions, herdtype, age categories and breeds. Herd prevalences, based on confirmation of single reactors, for dairy herds were estimated to be 16% (95%CI: 10-24%) in Sweden, 49% (95%CI: 39-59%) in Germany, 63% (95%CI: 57-69%) in Spain and 76% (95%CI: 67-84%) in The Netherlands and for beef herds 41% (95%CI: 31-50%) in Germany, 46% (95%CI: 41-51%) in Spain and 61% (95%CI: 50-72%) in The Netherlands. No beef herds were examined in Sweden. The lowest animal true prevalence was estimated in dairy cattle in Sweden (0.5% (95%CI: 0.1-0.8%)) while the highest animal true prevalence was estimated for dairy cattle in Spain (16.2% (95%CI: 14.9-17.5%)). Within-herd prevalences varied greatly, with very few farms in Sweden having more than 10% seropositive animals while in Spain more than 10% of the herds had within-herd prevalences between 50 and 100%. Seropositivity was significantly associated with herdtype (beef versus dairy), age, breed and region within countries. The results of this supranational comparative study showed that the importance of N. caninum infection varied greatly within in Europe. Estimates of prevalence can be used to calculate the economic impact of N. caninum infection as well as to evaluate the effect of prevention and control strategies over time.

Prevalence of Neospora caninum infection in Sardinian dairy farms (Italy) detected by iscom ELISA on tank bulk milk.

Neospora caninum is a heteroxenous cyst-forming coccidian closely related to Toxoplasma gondii and is considered one of the major causes of abortions in cattle worldwide. The present work aims to update the epidemiological trend of N. caninum of dairy cattle in Sardinia island, Western Mediterranean (Italy). For this reason, we used the newest enzyme-linked immunoassay (ELISA) methodology that exploits immune-stimulating complexes (iscoms) principle and allows us to point out the infection in the tank bulk milk too, besides the individual cattle. A total of 624 herds were sampled and tank bulk milk was submitted to iscom ELISA test. The analysis of the tank bulk milk samples revealed a total farm prevalence of 55% for N. caninum in Sardinia. In the provinces of Oristano and Cagliari the prevalences (64 and 65%, respectively) were significantly higher (p<0.01) than in Sassari and Nuoro (41 and 40%, respectively). The iscom Elisa test applied on tank bulk milk seems to be helpful and cost-effective for large epidemiological surveys, for monitoring control strategy plans for N. caninum, and for increasing the bio-safety level in dairy cattle farms.

HPLC purification of recombinant NcGRA6 antigen improves enzyme-linked immunosorbent assay for serodiagnosis of bovine neosporosis.

The gene for a dense granule protein (NcGRA6) of Neospora caninum was expressed in Escherichia coli as a His-tag fusion protein and purified by NiNTA affinity chromatography. In a preliminary study, high binding of antibodies from N. caninum-negative cows was observed in enzyme-linked immunosorbent assay (ELISA) using NiNTA-purified NcGRA6. Analysis of NiNTA eluates revealed a significant number of E. coli proteins that co-purified with recombinant NcGRA6. In an attempt to improve the relative sensitivity and specificity of the NcGRA6-based ELISA, the rNcGRA6 eluates were subjected to a secondary purification using reverse phase-high performance liquid chromatography (RP-HPLC). Analysis of RP-HPLC eluates by SDS-PAGE/silver staining revealed the purification of recombinant NcGRA6 from contaminating E. coli proteins. ELISAs using the RP-HPLC purified NcGRA6 (dELISA) or singly purified NcGRA6 (sELISA) for identifying seropositive and seronegative cows in a beef herd experiencing an epidemic outbreak of neosporosis were compared to standard assays based on native tachyzoite protein-immunofluorescence antibody test, immunoblot assay, and ISCOM-ELISA. The relative sensitivity, specificity, and kappa value of the NcGRA6d-ELISA were greatly improved over the NcGRA6s-ELISA when compared to the three native antigen immunoassays. These results indicate that removal of contaminating E. coli proteins improves the performance of recombinant NcGRA6 ELISA in diagnosing bovine neosporosis, and may have applicability to the use of recombinant proteins in diagnosing other infectious agents.

IgG avidity pattern in cattle after ingestion of Neospora caninum oocysts.

The avidity (functional affinity) of specific antibodies are being used to estimate duration of bovine Neospora caninum infection. Here, we report for the first time the avidity pattern in cattle orally inoculated with N. caninum oocysts. In all, 16 pregnant cows and 7 calves were administered N. caninum oocysts. In the cows, the avidity increased during the early course of infection. In all but one, the avidity was < or = 35 during the first 6 weeks after infection and no cow had an avidity value >50 until week 9. The calves were sampled either week 6 (n = 3) or week 9 (n = 9) after infection, and by then had avidities between 2 and 17. The results are in agreement with results from previous investigations of naturally infected cattle, and calves that were experimentally infected with tachyzoites. They further validate the ability of the N. caninum iscom avidity ELISA to accurately assess the duration of infection.

An interlaboratory comparison of immunohistochemistry and PCR methods for detection of Neospora caninum in bovine foetal tissues.

Seven European laboratories contributed to a multi-centre evaluation of detection techniques for Neospora caninum in bovine foetuses. Six laboratories participated in immunohistochemistry (IHC) testing. All seven laboratories participated in PCR testing, but the results from one laboratory were not included in the analysis, because of contamination problems in the preparation of the samples. A coded panel of tissue sections from 36 infected and non-infected foetuses was used to evaluate the IHC detection of parasites. A coded panel consisting of 44 homogenized foetal brain samples from natural bovine abortion cases and 32 spiked samples were used to evaluate the PCR methods. Inclusion of a duplicate dilution series of spiked samples was used to evaluate detection limits and repeatability. IHC methods had a relatively low sensitivity, but a high specificity. There was considerable variation in IHC results between participating laboratories, which may be partly explained by examination practices that depended on the experience of the operator. In addition, the use of different antibody reagents, different antibody dilutions, and different enzymatic treatments of tissues may have contributed to the observed variation. PCR methods generally had a higher sensitivity than IHC methods and also a high specificity. The agreement between the majority scores of IHC and PCR methods was low. False positive PCR results indicated contamination problems in some instances. Agreement between the PCR results of the various laboratories was better, compared with the IHC results. There appeared to be no clear relationship between the PCR format (i.e. single or nested) and diagnostic sensitivity. Consequently, an improvement of diagnostic performance of PCR might possibly be achieved by optimizing DNA extraction methods.

Toxoplasma gondii and Neospora caninum serological status of different canine populations from Uberlândia, Minas Gerais

Este trabalho teve como objetivo analisar a ocorrência de anticorpos contra Toxoplasma gondii e Neospora caninum em cães da cidade de Uberlândia, MG. As taxas de reatividade sorológica dos animais frente a esses parasitos foram comparadas em relação à sua origem e condições de vida. Um total de 369 amostras de soro canino foi obtido de três grupos de cães: (A) 213 pacientes do Hospital Veterinário da Universidade Federal de Uberlândia, (B) 62 pacientes de clínicas veterinárias particulares da cidade e (C) 94 cães errantes da cidade. As amostras foram analisadas para a presença de anticorpos contra os parasitos por meio de ELISA. Os resultados mostraram soropositividades de 30,3 por cento para T. gondii, 9,2 por cento para N. caninum e 5,7 por cento para ambos os parasitos. O grupo de cães errantes apresentou maior taxa de prevalência de anticorpos para os dois parasitos estudados. Pode-se concluir que as taxas de soropositividades para T. gondii e N. caninum nas populações caninas estudadas parecem ser fortemente influenciadas por suas origens e condições de vida.

Comparison and standardisation of serological methods for the diagnosis of Neospora caninum infection in bovines.

Various existing serological tests were compared with a standard panel of 523 sera in a multicentred study across Europe. Well characterised sera from animals that were experimentally or naturally infected with Neospora caninum as well as sera from cattle deemed uninfected with N. caninum were provided by the participants of the study and analysed in several commercial (CHEKIT Dr. Bommeli/Intervet, CIVTEST BOVIS NEOSPORA Hipra, Cypress Diagnostics C.V., Herd Check IDEXX, Mastazyme MAST Diagnostics, P38-ELISA Animal Welfare and Food Safety GmbH (AFOSA)) as well as in-house assays (five ELISAs and one IFAT). Most tests showed a high level of agreement in the interpretation of the test results (positive or negative). A further distinct increase in agreement between tests was obtained after the application of standardised cut-offs offered by a two-graph receiver operating characteristic analysis. This procedure allows a standardised interpretation of results obtained with different tests used in independent, parallel seroepidemiological studies.

Transplacental transmission and abortion in cows administered Neospora caninum oocysts.

Neospora caninum infection is a common cause of bovine abortion. One method by which cattle can acquire infection is through ingestion of oocysts; however, this has not yet been proved to cause transplacental infection or abortion. In this study, 19 cows, pregnant between 70 and 176 days, were administered 1500 to 115,000 oocysts through an esophageal tube. Seventeen of the cows became seropositive, indicating acquisition of infection, whereas 8 negative control cows remained seronegative (P < 0.001). Offspring were examined using serology, histology, immunohistochemistry, parasite isolation, and polymerase chain reaction (PCR). Six offspring were infected and 1 of them was aborted. The aborted fetus had typical lesions and positive immunohistochemistry and PCR for N. caninum. All 6 cows with infected offspring had continuously rising antibody titers, whereas 10 of 11 infected cows with uninfected offspring had falling titers after an early apex. The risk of transplacental transmission was increased by later exposure times during gestation and by the dose of oocysts (P < 0.01 for the 2 combined variables). The lowest dose of oocysts, when administered after the 160th day of gestation, caused transplacental infection in 1 of 2 animals. This study demonstrates that infection with N. caninum oocysts can cause transplacental transmission and abortion in cattle.