Prevalence and molecular characterization of Cryptosporidium spp. and Enterocytozoon bieneusi from large-scale cattle farms in Anhui Province, China

To investigate the prevalence of Cryptosporidium spp. and Enterocytozoon bieneusi from large-scale cattle farms in Anhui Province, 955 fecal samples were collected from 16 cattle farms from March to October 2018, which included six dairy farms (526), seven yellow cattle farms (323), and three water buffalo farms (106) in different regions of Anhui Province. PCR was conducted on all fecal samples using the 18S ribosomal RNA of Cryptosporidium spp. and internal transcribed spacer gene of E. bieneusi to detect these two pathogens, and the positive samples were sequenced and analyzed. The results showed that 23 (2.4%) and 40 (4.2%) out of the 955 samples were positive for Cryptosporidium spp. and E. bieneusi, respectively. There were 11 (2.1%), 10 (3.1%), and 2 (1.9%) positive samples of Cryptosporidium spp. and 16 (3.0%), 23 (7.1%), and 1 (0.9%) positive samples of E. bieneusi collected from dairy cattle, yellow cattle, and water buffalo, respectively, and no co-infection was identified in this study. All positive samples of Cryptosporidium spp. were C. andersoni with some variations. Ten E. bieneusi genotypes were obtained, including two known genotypes, J and CHN11, and eight new genotypes, named AHDC1 and AHYC1-7. The genotype CHN11 belonged to zoonotic Group 1, and the other nine genotypes belonged to Group 2, which is mainly documented in ruminants. These results indicated that Cryptosporidium spp. and E. bieneusi infections were present in large-scale cattle farms in Anhui Province. Therefore, attention should be paid to the development of containment strategies of these two pathogens in cattle.     

Prevalence,quantitative load and genetic diversity of Campylobacter spp. in dairy cattle herds in Lithuania

Background

Campylobacteriosis is a zoonotic disease, and animals such as poultry, pigs and cattle may act as reservoirs for Campylobacter spp. Cattle shed Campylobacter spp. into the environment and they can act as a reservoir for human infection directly via contact with cattle or their faeces or indirectly by consumption of contaminated food. The aim of this study was to determine the prevalence, the quantitative load and the genetic strain diversity of Campylobacter spp. in dairy cattle of different age groups.

Results

Faecal samples of 200 dairy cattle from three farms in the central part of Lithuania were collected and examined for Campylobacter. Cattle herds of all three farms were Campylobacter spp. positive, with a prevalence ranging from 75% (farm I), 77.5% (farm II) to 83.3% (farm III). Overall, the highest prevalence was detected in calves (86.5%) and heifers (86.2%). In contrast, the lowest Campylobacter prevalence was detectable in dairy cows (60.6%). C. jejuni, C. coli, C. lari and C. fetus subsp. fetus were identified in faecal samples of dairy cattle. C. upsaliensis was not detectable in any sample. The high counts of Campylobacter spp. were observed in faecal material of dairy cattle (average 4.5 log₁₀ cfu/g). The highest numbers of Campylobacter spp. were found in faecal samples from calves (average 5.3 log₁₀ cfu/g), whereas, faecal samples from cows harboured the lowest number of Campylobacter spp. (average 3.7 log₁₀ cfu/g). Genotyping by flaA PCR-RFLP analysis of selected C. jejuni isolates showed that some genotypes were present in all farms and all age groups. However, farm or age specific genotypes were also identified.

Conclusions

Future studies are needed to investigate risk factors related to the degree of colonisation in cattle. Based on that, possible measures to reduce the colonisation and subsequent shedding of Campylobacter in cattle could be established. It is important to further investigate the epidemiology of Campylobacter in the cattle population in order to assess associated risks to public health.     

Prevalence of species and genotypes of Cryptosporidium found in 1-2-year-old dairy cattle in the eastern United States

The prevalence of Cryptosporidium species in 1-2-year-old heifers was determined for 571 animals on 14 dairy farms in seven states on the East Coast of the United States. A fecal specimen collected directly from each heifer was processed to concentrate oocysts that were then examined by polymerase chain reaction (PCR). For every PCR-positive specimen the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified by PCR from heifers on 13 of 14 farms. On all except four farms groups of heifers were housed in a barn or in large covered pens. Others were pastured. From many of the same farms an earlier study reported that 41% of 393 pre-weaned calves and 26.2% of 447 post-weaned calves were infected. In the present study, 11.9% of 571 heifers were infected with Cryptosporidium, 0.7% with Cryptosporidium parvum, the zoonotic species. Of 68 PCR-positive specimens characterized by gene sequencing 1, 4, 10, 24, and 29 calves were infected with Cryptosporidium suis, Cryptosporidium parvum, Cryptosporidium deer-like genotype, Cryptosporidium bovis, and Cryptosporidium andersoni, respectively. These findings demonstrate a lower prevalence of infection in 1-2-year-old dairy cattle than in younger cattle as well as a change in the diversity of species present. Consequently, the risk of humans acquiring infection with C. parvum from exposure to feces from yearling and older cattle appears much lower than from exposure to pre-weaned calves.     

Abundance of biting midge species (Diptera: Ceratopogonidae,Culicoides spp.) on cattle farms in Korea

Culicoides biting midges were collected on three cattle farms weekly using light traps overnight from May to October between 2010 and 2011 in the southern part of Korea. The seasonal and geographical abundance of Culicodes spp. were measured. A total of 16,538 biting midges were collected from 2010 to 2011, including seven species of Culicoides, four of which represented 98.42% of the collected specimens. These four species were Culicodes (C.) punctatus (n = 14,413), C. arakawae (n = 1,120), C. oxystoma (n = 427), and C. maculatus (n = 318). C. punctatus was the predominant species (87.15%).     

Occurrence of Cryptosporidium and Giardia on beef farms and water sources within the vicinity of the farms on Prince Edward Island, Canada

The objectives of this study were to determine the prevalence and assemblages of Giardia and species of Cryptosporidium on beef farms in Prince Edward Island (PEI), Canada, including the water sources associated with the farms, and to determine risk factors for infection of cattle with these parasites. Twenty beef farms were selected based on the presence of surface water < 500 m from the barn. Prevalence was determined by direct immunofluorescence microscopy, while genotyping and species determination were performed by nested-PCR and DNA sequencing. Giardia was detected in 42% (95% CI: 38-46%) of fecal samples from 100% farms while Cryptosporidium was detected in 17% (95% CI: 14-19%) of fecal samples from 80% of farms. The most predominant Giardia assemblage isolated was the livestock specific assemblage E (89%). The zoonotic assemblages A and B were found in 4 and 7% of the Giardia isolates that were genotyped, respectively. The Giardia assemblages were detected equally between the cows and calves examined. Overall, the most common Cryptosporidium species detected in this study was Cryptosporidium andersoni (49%), predominantly found in cattle >6 mo of age, while most Cryptosporidium bovis and Cryptosporidium pestis (previously Cryptosporidium parvum ‘bovine genotype’) isolates were detected in calves ≤ 6 mo of age. All Cryptosporidium ryanae isolates (four) were found in calves. Giardia cysts and Cryptosporidium oocysts were detected in 14 and 93% of surface water samples of 14 farms, respectively. Cryptosporidium oocysts were detected in three (15%) ground water samples of 20 farms. One Cryptosporidium-positive water sample, which was the only surface water sample amenable to genotyping, contained C. parvum. The farm-level risk factors investigated in this study, age of animals and location of the farm, were not associated with the risk of infection in cattle with either Cryptosporidium spp. or Giardia duodenalis.We conclude that beef cattle are a potential reservoir of Cryptosporidium spp. and G. duodenalis that could contaminate source water. There is the possibility of further transmission to humans on PEI if the source water is not properly treated prior to consumption.     

Prevalence and molecular characterization of Giardia duodenalis and Cryptosporidium spp. in dairy cattle in Ontario, Canada

Giardia duodenalis and Cryptosporidium spp. are intestinal protozoan parasites that infect a wide range of host species, including humans. Molecular characterization of these parasites has demonstrated that a number of genotypes and species are common to both humans and animals, and that zoonotic transmission may occur. Numerous studies have reported a high prevalence of G. duodenalis and Cryptosporidium spp. in cattle, particularly calves, and these animals are frequently associated with zoonotic transmission. In the present study, a total of 143 faecal samples from adults, heifers and calves were collected from two dairy cattle farms in eastern Ontario, Canada. The prevalence and molecular characteristics of G. duodenalis and Cryptosporidium spp. in these animals were determined in order to investigate the potential for transmission between cattle and humans in this region. Following DNA extractions from faecal samples, nested-PCR protocols were used to amplify fragments of the 16S rRNA gene and the heat-shock protein 70 (HSP-70) gene for determining the prevalence of G. duodenalis and Cryptosporidium spp. infections, respectively. Genotypes of G. duodenalis, and species of Cryptosporidium, were determined by means of DNA sequencing of amplicons, and subsequent sequence alignment. Cattle on both farms showed a high prevalence of G. duodenalis (42.0%) and Cryptosporidium spp. (27.3%). G. duodenalis infections were more prevalent in calves and heifers than in adults, and Cryptosporidium spp. infections were only observed in calves and heifers. The zoonotic genotype, G. duodenalis Assemblage B was isolated from 24.5% of the cattle tested, while G. duodenalis Assemblage E was found in 17.5% of the cattle tested. The overall prevalence of the zoonotic species Cryptosporidium parvum in the animals tested was found to be 21.7%, while only 1.4% were infected with C. bovis. These findings suggest that there is a potential risk of zoonotic and/or zooanthroponotic transmission of G. duodenalis and C. parvum infections between cattle and humans in eastern Ontario, likely by means of contaminated water or food, or through direct faecal-oral transmission in the case of farmers and veterinary staff.     

Prevalence and molecular analysis of Sarcocystis infections in cattle in Northwest Iran and the first global report of S. gigantea in cattle

Cattle are intermediate host for several species of Sarcocystis, including S. cruzi, S. hirsuta, and S. hominis with high prevalence worldwide. The present study aimed to determine the prevalence of Sarcocystis infection, species identification, and phylogenetic analysis of the parasite in cattle in Northwest Iran. The samples of diaphragm and esophagus from 290 cattle were collected from slaughterhouses in Northwest Iran and subjected to macroscopic, microscopic, and histopathology examinations, PCR-RFLP, sequencing and phylogenetic analyses. Tissue cysts of Sarcocystis spp. were detected in 92% of cattle by digestion and microscopic tests. Based on the PCR–RFLP and specific PCR, 87.9% and 1.03% of isolates were identified as S. cruzi, and S. hominis, respectively. Macrocyst was seen in a single sample that was identified as S. gigantea. The haplotype network exhibited the extension of the various haplotypes of S. cruzi between neighboring provinces in Northwest Iran. Heterogeneity analysis of S. cruzi 18S-rRNA sequences indicated genetic diversity among S. cruzi isolates (Haplotype diversity: 0.733-0.854) consisting 16 haplotypes; however, the nucleotide differences showed low diversity (0.01481 to 0.03351). Pair wise sequence distance matrix amongst S. cruzi sequences indicated an intra-species divergence of 0%-7.8% and identity of 92.6%-100%. Sarcocystis infection is highly prevalent in cattle in Northwest Iran, with the predominance of S. cruzi, and genetic variants of this species are unequivocally distributing in Northwest provinces. First global detection of S. gigantea in cattle reflects new insights of transmission dynamic and biology of this parasite in Iran.     

Prevalence and genetic characterization of Giardia and Cryptosporidium in cats from Italy

One hundred and eighty one cats living in central Italy were tested for the presence of Giardia and Cryptosporidium infection by IFAT test and specific PCRs. Overall eight (4.4%) samples were IFAT-positive for Giardia. All the IFAT-positive samples for Giardia scored positive for the PCRs, and three more samples IFAT-negative generated PCR products leading to a total 6.1% molecular positivity rate for Giardia. All the examined samples were negative for Cryptosporidium. Sequencing of samples molecularly positive to Giardia indicated that three cats harbored the zoonotic Giardia duodenalis Assemblage A, whereas all other positive animals were infected with the feline-specific G. duodenalis Assemblage F. Phylogenetic analysis carried out on the sequences obtained supported the clustering of the isolates within Assemblages A and F. The results here presented provide data on the occurrence of Giardia genotypes in cats living in close contact with humans highlighting the potential importance of this protozoan disease for the public health.     

Occurrence and molecular characterization of Cryptosporidium spp. and Enterocytozoon bieneusi in dairy cattle,beef cattle and water buffaloes in China

Cryptosporidium spp. and Enterocytozoon bieneusi are important protists in a wide range of vertebrate hosts, causing diarrheal diseases. Cattle are considered potential reservoirs of Cryptosporidium infection in humans, although their role in the transmission of E. bieneusi is not clear. In the present work, 793 fecal specimens from dairy cattle, native beef cattle, and water buffaloes on 11 farms in China were examined for the presence of Cryptosporidium spp. and E. bieneusi using nested PCR targeting the small subunit (SSU) rRNA gene of Cryptosporidium spp. and the internal transcribed spacer (ITS) of E. bieneusi. For Cryptosporidium, 144/446 (32.3%) dairy cattle, 44/166 (26.5%) beef cattle, and 43/181 (23.8%) water buffaloes were PCR-positive. Sequence analysis was successful for 213 of the 231 Cryptosporidium-positive isolates; among them 94 had Cryptosporidium andersoni, 61 had Cryptosporidium bovis, 54 had Cryptosporidium ryanae, 2 had a Cryptosporidium suis-like genotype, and 2 had mixed infections of C. bovis and C. ryanae. In dairy and beef cattle, C. andersoni and C. bovis were the most common species, whereas C. ryanae was the dominant species in water buffaloes. The latter species produced SSU rRNA sequences different between cattle and water buffaloes. For E. bieneusi, the infection rate of E. bieneusi in dairy cattle, beef cattle and water buffaloes was 4.9%, 5.4% and 2.2%, respectively. All 35 E. bieneusi-positive specimens were successfully sequenced, revealing the presence of four genotypes: three Group 2 genotypes previously reported in cattle as well as humans (I, J and BEB4) and one Group 1 genotype recently reported in yaks (CHN11). Genotypes I and J were the most common genotypes in dairy and beef cattle, while genotype CHN11 was the only genotype seen in water buffaloes. Thus, the distribution of Cryptosporidium spp. and E. bieneusi in water buffaloes might be different from in dairy and beef cattle in China. These findings indicate that some of the Cryptosporidium species and all four E. bieneusi genotypes identified in bovine animals in the study areas may have zoonotic potential.     

Prevalence of Giardia spp. and Cryptosporidium spp. on dairy farms in southeastern New York state

A prevalence study was designed to determine the presence of Cryptosporidium spp. and Giardia spp. in the soil of 37 dairy farms in southeastern New York state. A sampling design was developed and used to collect soil samples from these farms. Areas on the farms which were considered to be potential sources of contamination to the environment were evaluated quantitatively using a multidimensional scale. This scale included factors which could have the potential to contribute to the risk of contamination of the environment with Giardia or Cryptosporidium. In addition, the runoff pathway from these areas was identified and sampling points along that pathway were determined. Using a sampling grid, sampling sites were determined and soil samples collected and analyzed individually for the following: presence of Giardia and Cryptosporidium, pH, gravimetric moisture content, and volumetric moisture content. Out of 782 soil samples, 17% were positive for Cryptosporidium and 4% were positive for Giardia. The pH of the soil ranged from 3.7 to 9.8 with a mean of 7.0. There was a significant association between the pH and the likelihood of detecting Cryptosporidium spp. As the pH increased, the likelihood of detecting an oocyst decreased. Gravimetric moisture content had a mean of 40% and a range from 7 to 86%. There was a significant association between the gravimetric moisture content and the likelihood of detecting Giardia in soil samples.     

Prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) from cattle in Korea between 2010 and 2011

A total of 156 Shiga-like toxin producing Escherichia coli (STEC) were isolated from fecal samples of Korean native (100/568, 18%) and Holstein dairy cattle (56/524, 11%) in Korea between September 2010 and July 2011. Fifty-two STEC isolates (33%) harbored both of shiga toxin1 (stx1) and shiga toxin2 (stx2) genes encoding enterohemolysin (EhxA) and autoagglutinating adhesion (Saa) were detected by PCR in 83 (53%) and 65 (42%) isolates, respectively. By serotyping, six STEC from native cattle and four STEC from dairy cattle were identified as O-serotypes (O26, O111, O104, and O157) that can cause human disease. Multilocus sequence typing and pulsed-field gel electrophoresis patterns highlighted the genetic diversity of the STEC strains and difference between strains collected during different years. Antimicrobial susceptibility tests showed that the multidrug resistance rate increased from 12% in 2010 to 42% in 2011. Differences between isolates collected in 2010 and 2011 may have resulted from seasonal variations or large-scale slaughtering in Korea performed to control a foot and mouth disease outbreak that occurred in early 2011. However, continuous epidemiologic studies will be needed to understand mechanisms. More public health efforts are required to minimize STEC infection transmitted via dairy products and the prevalence of these bacteria in dairy cattle.     

Field efficacy of minidosed eprinomectin against Hypoderma spp. in dairy cattle

A chemoprophylactic field trial was conducted to assess the efficacy of pour-on eprinomectin applied at the approximate dose of 50 mcg/kg to dairy cattle with naturally occurring hypodermosis. Two-hundred-eleven cattle, selected from two herds with a high prevalence of Hypoderma spp. infestation, were divided in three groups: Group A (N = 71) was treated with pour-on eprinomectin at the recommended dosage of 500 mcg/kg, Group B (N = 64) at the lower dose of 50 mcg/kg, a third group (Group C, N = 76) served as untreated control group. Treatments were performed in November-December 2002 and the animals were examined for the presence of warbles in the following April and June. No larvae emerged in the treated groups, whereas a variable number of warbles (ranging from 1 to 28) were found in control animals. Adverse reactions were not observed in any animal, and only minor side effects were observed. A larger field trial carried out in the following year (1064 treated and 131 untreated control cattle) confirmed the chemoprophylactic efficacy of minidosed eprinomectin against Hypoderma spp. Administration of eprinomectin minidoses in dairy cattle is interesting because of the low costs involved and no need for milk withdrawal.     

Prevalence of Cysticercus bovis in Australian cattle

Objective The first national abattoir survey of Cysticercus bovis (‘beef measles’) in cattle was conducted in February 2008. Methods During the data collection period, 493,316 cattle were subjected to standard postmortem procedures, including incision of the masseter and heart muscles. On-site veterinarians were asked to record the location of any C. bovis cysts, as well as the National Livestock Identification System ear tag numbers of infected animals. Veterinarians were asked to submit samples for laboratory confirmation by histology and polymerase chain reaction testing. Results Of the 23 samples submitted, none was positive for C. bovis by either diagnostic method. Conclusions Occasional, isolated diagnoses of beef measles are still made in most states of Australia, but since the last regional surveys were conducted 30 years ago, when the estimated prevalence was 50 to 200 per 100,000 cattle slaughtered, the parasite has become extremely rare.     

Epidemiology and genetic characterization of BVDV,BHV-1, BHV-4, BHV-5 and Brucella spp. infections in cattle in Turkey

The aim of the study was to determine the epidemiological data of bovine viral diarrhea virus (BVDV), bovine herpesvirus-1 (BHV-1), bovine herpesvirus-4 (BHV-4), bovine herpesvirus-5 (BHV-5) and Brucella–associated cattle that were previously reported to have abortion and infertility problems in Ankara, Corum, Kirikkale and Yozgat provinces, Turkey. Whole blood and sera samples were obtained from 656 cattle, and antibodies against Brucella spp. were detected in 45 (6.86%) and 41 (6.25%) animals by Rose Bengal plate and serum tube agglutination tests, respectively. The seropositivity rates against BVDV, BHV-1 and BHV-4 were 70.89%, 41.3% and 28.78%, respectively. RT-PCR and PCR were performed to detect RNA and DNA viruses in blood samples, respectively. The BVDV 5′-untranslated region and BHV-1 gB gene detected in this study were phylogenetically analyzed. The BVDV strains analyzed in this study were closely related to those previously reported from Turkey. The nucleotide sequence from the BHV-1 strain detected in this study is the first nucleotide sequence of BHV-1 circulating in this area of Turkey deposited in the GenBank. The presence of Brucella spp. and prevalence of BHV-1, BHV-4 and BVDV in cattle should be further investigated throughout these regions.     

Prevalence and geographical distribution of Giardia spp. and Cryptosporidium spp. in dairy farms in Québec.

Giardiasis and cryptosporidiosis are parasitic infections of humans, domestic animals, and wildlife. In cattle, direct transmission through the contamination of barns and/or pasture appears to be the principal mode of infection. In Canada there is only one study reported in the literature on the prevalence of giardiasis and one study on the prevalence of cryptosporidiosis. These studies were done in Alberta and Manitoba, respectively. The purpose of this study was to determine the prevalence of Giardia spp. and Cryptosporidium spp. infections in dairy farms in Québec. Calves were sampled from 505 dairy farms. We are reporting that 45.7% of the farms were found to be positive for Giardia spp. and 88.7% were infected with Cryptosporidium spp.     

Sporozoite proteome analysis of Cryptosporidium parvum by one-dimensional SDS-PAGE and liquid chromatography tandem mass spectrometry

Despite the development of new technologies, new challenges still remain for large scale proteomic profiling when dealing with complex biological mixtures. Fractionation prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis is usually the preferred method to reduce the complexity of any biological sample. In this study, a gel LC-MS/MS approach was used to explore the stage specific proteome of Cryptosporidium (C.) parvum. To accomplish this, the sporozoite protein of C. parvum was first fractionated using SDS-PAGE with subsequent LC-MS/MS analysis. A total of 135 protein hits were recorded from 20 gel slices (from same gel lane), with many hits occurring in more than one band. Excluding all non-Cryptosporidium entries and proteins with multiple hits, 33 separate C. parvum entries were identified during the study. The overall goal of this study was to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins present in lower abundance in a complex protein mixture.     

Characterization of coagulase negative staphylococci from cases of subclinical mastitis in dairy cattle in Kampala,Uganda

Background

Coagulase negative staphylococci (CNS) are the most common pathogens leading to subclinical mastitis (SCM) in dairy cattle in Uganda. Coagulase negative staphylococci can vary between bacterial species in how they cause disease. The aim of the study was to characterize CNS, from cows with SCM in Uganda, at the species level.

Findings

Quarter milk samples (n = 166) were collected from 78 animals with SCM. Bacteriological analyses were carried out at Makerere University, Kampala, Uganda and at the National Veterinary Institute (SVA), Uppsala, Sweden. The most common pathogens found in milk samples from cows with SCM were CNS (31.7%). Two species of CNS were found, S. epidermidis (85%) and S. haemolyticus (15%). Of the CNS isolates, 16/20 (80%) were positive for β-lactamase production (β+).

Conclusions

In milk samples from cows with SCM caused by CNS, S. epidermidis was most prevalent, followed by S. haemolyticus.     

Nationwide seroprevalence of Neospora caninum among dairy cattle in Japan

Serum samples from 2420 clinically healthy dairy cattle, randomly selected from stored sera in 18 districts of Japan, were tested for the presence of Neospora caninum antibodies using an indirect fluorescent antibody test (titer > or =1:200). Nationwide seroprevalence is estimated at 5.7% (139/2420). Seropositive cattle were detected in all surveyed districts despite the evidence of confirmed case reports of bovine neosporosis, showing that N. caninum is widely distributed throughout Japan. Age-specific seroprevalence did not increase with cattle age, suggesting that Neospora infection is likely to be transmitted vertically rather than horizontally in Japan. Considering that N. caninum seropositive cows are thought to be more likely to abort, substantial fetal losses may be induced by N. caninum infection in Japan. Devising strategies are needed to reduce the economic impact on the Japanese dairy industry. This is the first study to investigate the nationwide seroprevalence of N. caninum in cattle in Asia.