Prevalence and analysis of potential risk factors for Cryptosporidium parvum infection in lambs in Zaragoza (northeastern Spain)

An epidemiologic study was carried out to investigate the prevalence of and to identify factors associated with the risk of Cryptosporidium infection in sheep in Zaragoza (northeastern Spain). Faecal samples from 583 lambs aged from 1 day to 3 months and 205 ewes older than 1 year were collected at 89 farms in the two regions of the province of Zaragoza with the highest sheep population (Zaragoza and Ejea de los Caballeros). In every sheep farm, data of the factors potentially associated with the likelihood of C. parvum infection were analysed: geographical location, season, size of herd, number of lambs in the herd at sampling time, lambing period, cleaning of lambing area and presence of diarrhoeic lambs in the farm. C. parvum oocysts were identified by using the Ziehl-Neelsen technique in 344 lambs (59%) from 75 farms (84.4%). Infected lambs ranged from less than 7 days to 90 days of age, although the percentage of animals shedding oocysts peaked at 8-14 days of age (76.2%). Statistical analysis showed that infection rates were significantly higher in lambs aged between 1 and 21 days (66.4%) than in those aged between 22 and 90 days (23%) (P<0.0001, chi(2)). Analysis of correlation between excretion of oocysts and diarrhoea revealed a relationship in all age groups and the probability of presenting diarrhoea was significantly higher for lambs shedding oocysts (86.3%) than for those which did not excrete the parasite (32.2%) (P<0.0001, chi(2)). Similarly, cryptosporidial infection rates were significantly higher in diarrhoeic (79.4%) than in non-diarrhoeic lambs (22.4%). Furthermore, infection intensity was correlated with the presence of clinical symptoms. Presence of diarrhoeic lambs in the farm was the only factor significantly associated with an increased risk of infection since the percentage of herds testing positive was significantly higher in farms with diarrhoeic lambs (91.3%) than in those without cases of neonatal diarrhoea (12.5%) (P<0.0001, chi(2)). Factors associated with a decreased risk of C. parvum infection in lambs included low numbers of lambs in the farm and cleaning of the lambing area. Additionally, lambs 8-14 days of age were less likely to be infected at the first lambing period and in spring/autumn. Cryptosporidial infection was also detected in 16 ewes (7.8%) which excreted few oocysts and without diarrhoea.     

Cryptosporidium parvum infection in orphan lambs on a farm open to the public

A longitudinal survey was undertaken on an open farm to investigate the occurrence of Cryptosporidium species infection in orphan lambs obtained from three local flocks. During an initial pilot study, Cryptosporidium oocysts were detected by a fluorescent antibody test (fat) in the faeces of two of 21 lambs aged between one and three weeks derived from one flock (flock A). Pooled pen samples of faeces were collected weekly from lambs derived from each flock; oocysts were detected by fat in 24 (49.0 per cent) of 49 samples from lambs from flock A, 18 (30.5 per cent) of 59 samples from lambs from flock B and 14 (29.8 per cent) of 47 samples from lambs from flock C. Oocyst counts of 1 x 10(3) to more than 2 x 10(6) per gram of faeces were detected in lambs up to 12 weeks old, with the peak counts occurring at six weeks of age in the lambs from flocks A and B and at four weeks of age in those from flock C. The oocysts were confirmed by molecular analysis as Cryptosporidium parvum. Virtually all the infections were subclinical.     

Molecular characterization of Cryptosporidium isolates from pigs in Zaragoza (northeastern Spain)

A total of 142 stool specimens from pigs on 24 farms from the province of Zaragoza (northeastern Spain) were screened for Cryptosporidium spp. Samples were first analysed by routine techniques (formalin-ethyl acetate sedimentation method and modified Ziehl-Neelsen stain) selecting those microscopically positive for genetic characterization. Cryptosporidium species and genotypes were determined by a nested PCR-RFLP technique at the 18S ribosomal DNA locus and sequencing of the PCR-positive secondary products. Cryptosporidium oocysts were microscopically identified in the faeces of 32 pigs (22.5%) from 15 farms (62.5%). Infected animals included 23 weaned piglets (30.7%), 5 fattening pigs (11.9%) and 4 sows (16%). Diarrhoea was not detected in any of the infected pigs. The molecular characterization was successfully performed in 26 samples from 14 farms. Cryptosporidium suis was found in 10 specimens from 7 farms (nine weaned piglets and one sow) and the Cryptosporidium pig genotype II in 16 samples from 10 farms (13 weaned piglets and 3 fattening pigs). Both C. suis and the pig genotype II were concurrently detected on three farms.     

Cryptosporidium parvum in cattle, sheep and pigs in Galicia (N.W. Spain).

Infection by Cryptosporidium parvum has been detected for the first time in cattle and swine in Galicia (N.W. Spain). The organisms were also found in one of 69 sheep examined. Although in most cases the parasite was found in young diarrhoeic animals, its presence in asymptomatic mature cattle and piglets was also detected.     

Production and use of murine monoclonal antibodies reactive with bovine IgM isotype and IgG subisotypes (IgG1, IgG2a and IgG2b) in assessing immunoglobulin levels in serum of cattle

Utilizing in-vitro and in-vivo immunizations, murine monoclonal antibodies (Mab's) that are specific for bovine immunoglobulin (IG)--IgM isotype and IgG subisotypes (IgG1, IgG2a and IgG2b)--have been produced. These Mab's were used to estimate the quantities of these bovine Ig isotype and IgG subisotypes in the sera of 56 cows and 24 bulls. The cows were all approximately 18 months of age and the bulls ranged from 12 to 18 months in age. The cows and bull were all apparently healthy and unimmunized. The cows were not pregnant and were nulliparous. Some differences were observed between the serum levels of IgM (higher in cows) and IgG1 (higher in bulls). No ready explanation can be offered for these observed differences.     

Resistance patterns and detection of aac(3)-IV gene in apramycin-resistant Escherichia coli isolated from farm animals and farm workers in northeastern of China

The aminoglycoside apramycin has been used widely in animal production in China since 1999. This study was aimed to investigate the resistance pattern of apramycin-resistant Escherichia coli isolated from farm animals and farm workers in northeastern of China during 2004–2007 and to determine whether resistance to apramycin was mediated by plasmid containing the aac(3)-IV gene and the mode for the transfer of genetic information between bacteria of farm animals and farm workers. Thirty six E. coli isolates of swine, chicken, and human origins, chosen randomly from 318 apramycin-resistant E. coli isolates of six farms in northeastern of China during 2004–2007, were multi-resistant and carried the aac(3)-IV gene encoding resistance to apramycin. Conjugation experiments demonstrated that in all 36 cases, the gene encoding resistance to apramycin was borne on a mobilisable plasmid. Homology analysis of the cloned aac(3)-IV gene with the sequence (accession no. X01385) in GenBank showed 99.3% identity at a nucleotide level, but only with a deletion of guanosine in position 813 of the gene in all 36 cases. The results indicted that resistance to apramycin in these isolates was closely related to aac(3)-IV gene. Therefore, the multi-resistance of E. coli could complicate therapeutic practices for enteric infections in both farm animals and human.     

Prevalence of Giardia sp. Cryptosporidium parvum and Cryptosporidium andersoni (syn. C. muris) [correction of Cryptosporidium parvum and Cryptosporidium muris (C. andersoni)] in 109 dairy herds in five counties of southeastern New York

A cross-sectional study was undertaken to determine the prevalence of Giardia sp. (G. duodenalis group), Cryptosporidium parvum and Cryptosporidium andersoni (C. muris) [corrected] in dairy cattle in three different age groups, and to evaluate the association of age and season with prevalence. One hundred and nine dairy farms, from a total of 212 farms, in five counties of southeastern New York volunteered to participate. On these farms, 2943 fecal samples were collected from three defined age groups. The farms were randomly assigned for sampling within the four seasons of the year. Each farm was visited once during the study period from March 1993 to June 1994 to collect fecal samples. Demographic data on the study population was collected at the time of sampling by interviewing the farm owner or manager. At collection, fecal samples were scored as diarrheic or non-diarrheic, and each condition was later related to positive or negative infection with these parasites. Fecal samples were processed using a quantitative centrifugation concentration flotation technique and enumerated using bright field and phase contrast microscopy. In this study, the overall population prevalence for Giardia sp. was 8.9%; C. parvum, 0.9%; and C. muris, 1.1%. When considering animals most at the risk of infection (those younger than 6 months of age) Giardia sp. and C. parvum was found in 20.1 and 2.4% of the animals, respectively. Giardia sp. and C. muris were found in all age groups. There was no significant seasonal pattern of infection for any of these parasites.     

Recombinant bovine interleukin-12 stimulates a gut immune response but does not provide resistance to Cryptosporidium parvum infection in neonatal calves

This study was undertaken to determine if administration of recombinant bovine interleukin-12 (rBoIL-12) could stimulate a cellular immune response that protected calves from an oral challenge inoculation with Cryptosporidium parvum oocysts. In a first experiment, rBoIL-12 intraperitoneally administered as a single dose 1 day before challenge inoculation, did not alter the course of infection. The percentage of immune competent cells and levels of cytokine gene expression in the ileo-cecal mucosa and in the draining lymph nodes of treated calves were similar to those of untreated control calves. However, when rBoIL-12 was subcutaneously administered daily from 2 days before infection to 2 days after infection, a consistent increase of T lymphocytes and an higher expression of interferon-gamma (IFN-gamma) was detected. Again, treatment did not alter the course of infection. Similar results were obtained when rBoIL-12 was administered daily for 4 days beginning 2 days after oral inoculation. These data indicate that although rBoIL-12 stimulated a strong immune response in the gut of neonatal calves, the response was not able to provide protection from challenge inoculation with C. parvum oocysts.     

Interaction of bovine immunoglobulin gamma 2 (IgG2) with staphylococcal protein A: evidence for IgG2a and IgG2b sub-subclasses in the bovine

The reactivity of bovine IgG with protein A is confusing with respect to which of the bovine IgG class and subclasses are reactive. We have, therefore, re-examined the interaction of bovine immunoglobulins with protein A. The results presented in this paper indicated that at pH 8.0 protein A binds only immunoglobulin of the IgG2 subclass. The bound IgG2 can be readily recovered from an immobilized protein A column at pH 5.0. Furthermore, the antigenic IgG2 eluted demonstrated two charged species which could readily be separated by ion-exchange chromatography. These results indicate that IgG2 in the bovine exists in two sub-subclasses, IgG2a and IgG2b. The two sub-subclasses of IgG2 could be rapidly isolated with a good yield in two-steps namely protein A affinity chromatography followed by ion exchange chromatography.     

Serum levels of the immunoglobulins IgG and IgG(T) in horses

Levels of the immunoglobulins IgG and IgG(T) in serum in Norwegian horses of the breeds “Døle” and “Fjord” were determined by the quantitative radial immunodiffusion test.No significant differences were apparent between the 2 Norwegian breeds. The immunoglobulin levels were approximately in the same range as previously reported for Shetland ponies.Immunoglobulins could not be detected in the newborn foal. As early as 24 hrs. after birth the mean immunoglobulin level was within the adult range. After a drop during the first month of life, the immunoglobulins increased. IgG(T) rose more rapidly and to a higher level than IgG.In 2 year old horses, IgG(T) was significantly higher than in adults, while IgG was significantly lower. IgG(T) seems to be a very important immunoglobulin in foals and young horses.     

Attempts to protect severe combined immunodeficient (scid) mice with antibody enriched for reactivity to Cryptosporidium parvum surface antigen-1

Cryptosporidium paruum is a protozoal pathogen which infects the gastrointestinal epithelium of mammals causing diarrhoea, the duration and severity of which is determined by the immunocompetency of the host. Currently, there is no effective treatment or prevention. We evaluated the ability of surface antigen-1 (SA-1), defined as those antigens recognized by neutralizing mAb 17.41, to elicit a protective antibody response when used as an immunogen. A SA-1 enriched fraction was obtained by immunoaffinity chromatography and was used to immunize a naive Holstein calf. SA-1 immune serum from this calf detected C. parvum epitopes to a 1:10 000 dilution in a dot blot assay, and sporozoite surface epitopes at a 1:10 000 dilution in a live immunofluorescence assay. Western blot analysis showed that SA-1 immune bovine serum recognized a similar pattern of C. parvum antigens as the defining mAb 17.41. Oral passive transfer of SA-1 immune bovine serum did not protect severe combined immunodeficient (scid) mice or suckling BALB /c mice from initial infection with C. paruum, or terminate a persistent infection in scid mice.     

Cryptosporidium parvum Caused a Large Outbreak Linked to Frisée Salad in Finland, 2012

Over 250 individuals fell ill in five outbreaks caused by Cryptosporidium parvum in Finland, October–November 2012. The cases were connected by lunch meals at restaurants in four different cities. In two outbreaks, the same C. parvumIIdA17G1 subtype was found in patients’ stool samples which supports a single source of infection. Frisée salad was the only common food item served at the restaurants, and consumption of lunch salad containing the frisée salad was associated with the illness. Lunch customers who responded that they had eaten lunch salad were three times more likely to have become ill than those who had not answered whether they had eaten the salad or not (RR 2.66; 95% Cl 1.02–6.9, P‐value <0.01). Cryptosporidiosis should be considered as a causal agent in long‐lasting watery diarrhoea combined with abdominal cramps, and clinical samples should be tested for Cryptosporidium at the same time bacteria and viruses are tested. Measures to prevent contamination of ‘ready‐to‐eat vegetables’ with Cryptosporidium oocysts and methods to test frozen food samples should be developed.     

Molecular identification of Cryptosporidium parvum and Giardia duodenalis in the Italian water buffalo (Bubalus bubalis)

Livestock are commonly infected with protozoan parasites of the genera Cryptosporidium and Giardia, and some of the species and genotypes found in these animals have zoonotic significance. We characterized isolates of both parasites recovered from the Italian water buffalo (Bubalus bubalis), an economically important species whose milk is used for the production of "buffalo mozzarella" fresh cheese. Molecular analysis of the Cryptosporidium small subunit ribosomal DNA gene and of the Giardia beta-giardin gene shows the presence of both zoonotic parasites (Cryptosporidium parvum and Giardia duodenalis assemblage A) and host-specific parasites (G. duodenalis assemblage E), suggesting that water buffaloes can contribute to environmental contamination with oocysts and cysts potentially infectious to humans if their faeces are improperly disposed of. On the other hand, mozzarella cheese is probably a safe product, given that its production involves the treatment of cheese curd at 85-95 degrees C, which is likely to kill or inactivate the parasites.     

Prevalence of Cryptosporidium and Giardia in roe deer (Capreolus capreolus) and wild boars (Sus scrofa) in Galicia (NW, Spain)

Faecal samples from 224 roe deer (Capreolus capreolus) and 381 wild boars (Sus scrofa) shot during the 2008-2009 hunting season (August-January) in Galicia (NW Spain) were examined to determine the presence and intensity of infection by Cryptosporidium and Giardia. Analysis of a single sample from each of the roe deer revealed that the prevalence of cryptosporidiosis and giardiosis was 1.3% and 5.3% respectively. The prevalence of Giardia infection was significantly higher in juvenile female roe deer than in adult females, but no other significant differences were found in relation to age and sex. In wild boars, the prevalence of cryptosporidiosis and giardiosis was 7.6% and 1.3% respectively. The prevalence of Cryptosporidium infection was significantly higher in juvenile male wild boars than in adult males, but no other significant differences were found in relation to age or sex. In both groups of wild animals, the number of Cryptosporidium oocysts per gram of faeces (OPG) ranged from 5 to 200 and the number of Giardia cysts per gram of faeces (CPG) was between 5 and 47; there were no significant differences between the two groups with respect to number of infections. This is the first large study of Cryptosporidium and Giardia in roe deer and wild boars in hunting areas in Spain and the results demonstrate a low, but widespread prevalence of Cryptosporidium and Giardia in these animals.     

Comparison of an enzyme-linked immunosorbent assay and a complement-fixation test for the detection of IgG to bovine herpesvirus type 4 (bovine cytomegalovirus)

An indirect ELISA for the detection of bovine immunoglobulin to bovine herpesvirus type 4 (BHV-4) was developed. Three methods of antigen preparation were compared. They included (1) BHV-4-infected Madin Darby bovine kidney (MDBK) cells treated with glycine and then frozen and thawed, (2) BHV-4-infected MDBK cells treated with glycine and then sonicated, and (3) BHV-4-infected MDBK cells treated with a detergent. The antigen preparation that gave the highest reactivity was the first method. We obtained serum samples from 178 cattle in the field and assayed the serum by ELISA and the complement-fixation (CF) test. Eighty-six percent (153) of the serum samples were positive by ELISA, and 70% (124) were positive by the CF test. The ELISA had a higher degree of sensitivity than did the CF test. Also, the ELISA was specific, and the prevalence of BHV-4-infection was more common in beef and dairy herds than previously recognized.     

A novel multiplex polymerase chain reaction approach for detection of four human infective Cryptosporidium isolates: Cryptosporidium parvum, types H and C, Cryptosporidium canis, and Cryptosporidium felis in fecal and soil samples.

A nested multiplex polymerase chain reaction (PCR) approach was adopted for the simultaneous detection of 4 human infective genotypes of the protozoan parasite Cryptosporidium. Specific PCR primers were designed for the heat shock protein 70 gene of 2 genotypes of Cryptosporidium parvum (human and bovine types), Cryptosporidium canis, and Cryptosporidium felis. These 4 genotypes have all been found in human fecal samples. The primers amplified DNA fragments of specific sizes, each representing a unique genotype. The limit of detection of the method was found to vary between 10 and 100 oocysts per 1 ml fecal material. There appeared to be no cross-reactivity with other organisms commonly present in feces and soil, and the approach has a high specificity. The rapid identification of various human infective Cryptosporidium isolates is a part of the authors' long-term aim of determining the routes of infection with oocysts and thereby increase their epidemiological understanding of Cryptosporidium infection in humans and animals.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody (IgG) to Pasteurella haemolytica

The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.     

Resistance patterns and detection of aac(3)-IV gene in apramycin-resistant Escherichia coli isolated from farm animals and farm workers in northeastern of China

The aminoglycoside apramycin has been used widely in animal production in China since 1999. This study was aimed to investigate the resistance pattern of apramycin-resistant Escherichia coli isolated from farm animals and farm workers in northeastern of China during 2004–2007 and to determine whether resistance to apramycin was mediated by plasmid containing the aac(3)-IV gene and the mode for the transfer of genetic information between bacteria of farm animals and farm workers. Thirty six E. coli isolates of swine, chicken, and human origins, chosen randomly from 318 apramycin-resistant E. coli isolates of six farms in northeastern of China during 2004–2007, were multi-resistant and carried the aac(3)-IV gene encoding resistance to apramycin. Conjugation experiments demonstrated that in all 36 cases, the gene encoding resistance to apramycin was borne on a mobilisable plasmid. Homology analysis of the cloned aac(3)-IV gene with the sequence (accession no. X01385) in GenBank showed 99.3% identity at a nucleotide level, but only with a deletion of guanosine in position 813 of the gene in all 36 cases. The results indicted that resistance to apramycin in these isolates was closely related to aac(3)-IV gene. Therefore, the multi-resistance of E. coli could complicate therapeutic practices for enteric infections in both farm animals and human.