Incidence of and risks associated with Giardia infections in herds on dairy farms in the New York City Watershed

Background

The primary aims of this study were to determine the incidence of Giardia infections in dairy herds on farms in the New York City Watershed region and to evaluate risk factors associated with infections. Because co-infections of Giardia and Cryptosporidium spp. are common in this population, we also evaluated the effect of herd infection status on Giardia infections.

Methods

Farms were grouped into three cohorts based on their prior infection status with Giardia and/or Cryptosporidium spp. The sampling plan included collecting fecal samples from all calves below 30 days of age and proportional sampling of calves, young stock, and adults. A total of 10,672 fecal samples were collected and analyzed for the presence of Giardia cysts using zinc sulfate flotation. Herds enrolled in the study were sampled seasonally for a study period of two years. The probability of shedding cysts past a certain age and the factors that influenced the likelihood of shedding were evaluated using survival analysis. Linear regression was used to evaluate factors that were associated with the intensity of shedding.

Results

The majority of Giardia infections occurred in calves within their first 180 days of age, with the most number of calves shedding Giardia cysts between 11 and 20 days of age. The incidence of shedding of Giardia cysts ranged from 0.0004 per animal day for cattle in the low risk cohort to 0.0011 per animal day for cattle in the high risk cohort. The likelihood of shedding was influenced by the prior infection status of the herd and the season of collection. Infected animals shed on average 9,658 cysts/gram and the intensity of shedding Giardia cysts varied significantly with the age (p < 0.0001) and the season of collection (p = 0.0151 for Spring).

Conclusion

Giardia infections are common in dairy herds in the New York City watershed, particularly in calves less than 6 months of age. Seasonality may be an important factor in the perpetuation of infections based on changes in management practices corresponding to weather patterns of a particular season. A dairy herd''s prior infection status with Cryptosporidium influences the likelihood of infection with Giardia.     

Prevalence of Giardia duodenalis genotypes in adult dairy cows

The prevalence of Giardia duodenalis genotypes was determined in adult dairy cows. Fecal specimens were collected from two farms each in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Specimens, cleaned of fecal debris and concentrated using CsCl density gradient centrifugation, were subjected to PCR and DNA sequence analysis. The prevalence of G. duodenalis infection, ranged from 3% to 64%, with an average prevalence of 27% (144 positive cows out of 541 examined). DNA sequence analysis of the 16S rRNA gene revealed the presence of both Assemblage A and Assemblage E, G. duodenalis. Overall, Assemblage E was present in 25% of all animals tested and Assemblage A was present in 2% of the animals. As a percentage of G. duodenalis isolates, Assemblage E represented 94% and Assemblage A represented 6%. Although, most of the cows were infected with a genotype that is not known to be infectious for humans, adult cows on five farms did harbor varying levels of zoonotic Assemblage A, G. duodenalis. Therefore, although adult cows do not appear to be a significant source of human infectious cysts in the environment, the risk from this age group should not entirely be discounted.     

Prevalence of Giardia duodenalis genotypes in pre-weaned dairy calves

To determine the prevalence of Giardia genotypes in pre-weaned dairy calves, fecal samples were collected from a minimum of 18, 1-7-week-old dairy calves per farm on two farms each in the states of Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Samples cleaned of fecal debris and concentrated using CsCl density gradient centrifugation were stained and examined by immunofluorescence microscopy and also subjected to PCR and gene sequence analysis. Prevalence by PCR ranged from 9% on a farm in Pennsylvania to 93% on a farm in Vermont, with an average prevalence for 407 calves on 14 farms of 40%. Gene sequence analysis of the TPI, beta-giardin and 16S rRNA genes revealed 85% of the positive samples to be Assemblage E, while 15% were Assemblage A, although the percentages of these genotypes varied greatly from farm to farm. Some farms had no Assemblage A Giardia. Thus, while a majority of the calves were infected with a genotype that is not known to be infectious for humans, calves on 7 of 14 farms did harbor Assemblage A Giardia. Calves should be considered as a potential source of human infectious cysts in the environment.     

Prevalence and genotypes of Giardia duodenalis in post-weaned dairy calves

To determine the prevalence of Giardia genotypes in post-weaned dairy calves, fecal specimens were collected from 3 to 11-month-old dairy calves per farm on two farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Specimens cleaned of fecal debris and concentrated using CsCl density gradient centrifugation were stained and examined by immunofluorescence microscopy and also subjected to PCR and DNA sequence analysis. Overall, PCR provided more sensitive detection than IFA. Prevalence of Giardia infection, as detected by PCR ranged from 20% on NC-2 to 81% on VT-2, with an overall prevalence of 52% (237 positive samples out of 456 total). DNA sequence analysis of the 16S rRNA gene revealed 87% of the 237 Giardia isolates were Assemblage E, and 13% were Assemblage A although the prevalence of these genotypes varied greatly from farm to farm, with five farms having no Assemblage A Giardia. Therefore, Assemblage E was present in 45% of all animals tested and Assemblage A was present in 7% of the animals. Thus, while many of the calves were infected with a genotype that is not known to be infectious for humans, post-weaned calves on nine of 14 farms did harbor Assemblage A Giardia. Therefore calves should be considered as a potential source of human infectious cysts in the environment, with some farms representing a much higher risk than others.     

Prevalence of Giardia duodenalis assemblages in weaned cattle on cow-calf operations in the United States

To determine the prevalence of Giardia duodenalis in weaned beef calves on cow-calf operations in the United States, fecal specimens were collected from 819 calves (6-18 months of age) from 49 operations. After cleaning and concentration procedures to maximize recovery of cysts from feces, DNA was extracted from each of the 819 specimens. The presence of G. duodenalis was determined by nested PCR of a fragment of the SSU rRNA gene. All positive PCR products were subjected to sequence analysis. The overall sample level prevalence of Giardia was 33.5% with prevalence ranging from 0 to 100% among operations. The highest within herd prevalence of infected beef calves was found in one cow-calf operation from the South region (100%), followed by a cow-calf operation from the West region (90%), and three cow-calf operations from the Midwest region (87.5, 85, and 85%). Giardia was not detected in samples from 7 operations including 5 cow-calf operations from the South region, and 1 cow-calf operation each from the Midwest and West regions. Molecular analysis of the Giardia-positive samples identified assemblage E (or E-like) in 31.7% of all samples (260/819) and assemblage A in 1.2% (10/819). A mixed infection with assemblages A and E was observed in four calves from an operation in Midwest region. The potentially zoonotic assemblage A was detected in specimens from four operations in Midwest region. These findings indicate that most G. duodenalis found in weaned beef calves was assemblage E which represents no known zoonotic threat. However, the presence of assemblage A in a small number of animals poses a potential risk of infection to humans.     

Bovine herpes mammillitis in two New York dairy herds

Bovine herpes mammillitis was diagnosed in 2 New York dairy herds. Lesions ranged from vesiculation and ulceration of large (up to 10 cm wide) areas of udder and teat skin to single small (2-3 cm wide) plaques of edema. Some lesions resembled "umbilicated pocks" characteristic of cowpox virus infections. Recently freshened heifers were the most severely affected; older cows and heifers with less turgid udders had milder lesions. In 2 cows, incurable mastitis developed. In other cows, the lesions healed by centripetal growth of epidermis into the lesions. Diagnosis was made by isolation of bovid herpesvirus 2 from lesions in both herds and by serum-neutralization testing. Virus isolated from a cow in 1 herd was injected into 9 members of the same herd and may have been responsible for the absence of lesions on these animals; all other members of the herd were affected.     

Incidence of Cryptosporidium parvum in the dairy cattle population in a New York City Watershed

A longitudinal study of 2-year duration was conducted to determine the risk, as measured by incidence rate, of Cryptosporidium parvum infection among dairy cattle in the Catskill/Delaware Watershed of New York City (NYC), and the factors that predispose animals to the likelihood of infection. A proportional sampling scheme with follow up at quarterly farm visits was employed for heifers and cows. Additionally, all calves born on the 39 study farms were sampled once during the first four weeks of life and at least once more before weaning. Samples were analyzed for the presence of C. parvum using a quantitative centrifugation concentration flotation technique and a C. parvum-specific enzyme-linked immunosorbent assay (ELISA). Of the 9914 fecal samples collected, 747 were found to contain C. parvum. The average number of oocysts detected was 1.3x10(5)/g (range: 1.0/g--8.2x10(6)/g). The average age at time of first detection of the organism was 15.0 days with a standard deviation of 6.59 days. The age range of animals infected with C. parvum in the study population was 3--60 days (inclusive). The unadjusted (crude) incidence rate of C. parvum among the entire study population was 2.05 per 1000 animal-days. The unadjusted incidence rate among pre-weaned calves was 15.55 per 1000 animal-days. After controlling for age and prior protozoal risk level, no seasonal impact on the incidence of C. parvum was detected among animals less than 61 days by negative binomial regression. A seasonal impact was identified among the oocyst counts of infected animals after controlling for age and prior protozoal risk level.     

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 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 characterization of Giardia duodenalis in dogs in Israel

Three hundred and two stool samples were collected from municipal shelters and owned dogs in different geographical locations in Israel from December 2016 to September 2017 and examined for Giardia and assemblage type by PCR targeting the 18S rRNA and β-giardin genes. Overall Giardia prevalence was 24.5 % (74/30). Giardia prevalence was 1.9-fold higher in dogs ≤ 6 months old compared to > 6 ≤ 12 months old and older dogs [25/61 (41 %), 18/73 (24.6 %) and 31/166 (18.7 %), respectively, (p = 0.001)], 2.3-fold higher in winter [32/90 (35.5 %)] compared to its prevalence during autumn [15/60 (25 %)], spring [10/62 (16.1 %)] and summer [17/89 (19.1 %), p = 0.003)], and 2.7-fold more frequent among diarrheic dogs [23/43 (53.4 %)] compared to those with formed stools [51/253 (20.1 %)], (p = 0.001)]. The Giardia sp. assemblages detected were C and D. Higher infection rates in young, diarrheic dogs, sampled during winter, and housed in municipal shelters, indicates the need for targeted preventive measures.     

Prevalence and risk factors of Giardia duodenalis in dogs from Romania

The protozoan Giardia duodenalis is a mammalian-infecting parasite that produces diarrhoea and malabsorption in its hosts. A survey to investigate canine infections with G. duodenalis in Romania was undertaken between June 2008 and December 2009. The objectives of the study were to (i) estimate the prevalence of infection in different dog populations (kennels, shelters, shepherd, household) using microscopy and a commercially available enzyme-linked immunosorbent assay (ELISA) test kit; (ii) to establish the level of agreement and characteristics of the tests; and (iii) to identify risk factors for infection by multivariate logistic regression models. Faecal samples were collected from 614 dogs aged from 1 month to 16 years (mean ± SD=2.88 ± 2.86 years). Each sample was tested for the presence of cysts using a flotation method with saturated sodium chloride solution and 416 out of 614 stool samples were further examined for the presence of G. duodenalis specific antigens using Giardia Microwell ELISA (SafePath? Laboratories). Giardia cysts were identified in 8.5% of total dogs (52/614) and statistical significantly more frequently in dogs living in communities. The cysts prevalence according with dog populations was as follows: 7.2%(9/125) in kennel dogs; 16.5%(27/164 in shelter dogs; 4.3%(2/46) in shepherd dogs; 4.8%(4/84) in household dogs from urban areas; and 5.1%(10/195) in household dogs from rural areas. The overall prevalence of Giardia infection by ELISA was 34.6% (144/416). The prevalence was significantly higher in kennel dogs (50%; 13/26), shelter dogs (47.7%; 74/155) and shepherd dogs (40.5%; 17/42) than in household dogs from urban areas (34.1%; 15/44) and household dogs from rural areas (16.8%; 25/149). It was noticed poor agreement between microscopy and ELISA (k=0.19). The microscopy performed best, with an Youden Index of 0.74, a Se of 73.68% and a Sp of 100%. ELISA had 100% Sp, but only 19.44% Se. Young dogs (up to 12 months age) and living in communities were identified as risk factors for infection by multivariate logistic regression analysis. 71.2% (37/52) Giardia cysts positive dogs presented co-infections with other intestinal parasites: Toxocara canis (14/52; 26.9%), Isospora ohioensis (12/52; 23.1%), Ancylostoma caninum (9/52; 17.3%), Uncinaria stenocephala (7/52; 13.5%), Trichocephalus vulpis (6/52; 11.5%), Hammondia heydorni/Neospora caninum (5/52; 9.6%), Sarcocystis spp. (5/52; 9.6%), Isospora canis (4/52; 7.7%), Capillaria aerophila (3/52; 5.8%), Strongyloides stercoralis (2/52; 93.8%), Dipylidium caninum (1/52; 1.9%) and Toxascaris leonina (1/52; 1.9%).     

Molecular evidence for zoonotic transmission of Giardia duodenalis among dairy farm workers in West Bengal, India

No study in the past has examined the genetic diversity and zoonotic potential of Giardia duodenalis in dairy cattle in India. To assess the importance of these animals as a source of human G. duodenalis infections and determine the epidemiology of bovine giardiasis in India, fecal samples from 180 calves, heifers and adults and 51 dairy farm workers on two dairy farms in West Bengal, India were genotyped by PCR-RFLP analysis of the β-giardin gene of G. duodenalis followed by DNA sequencing of the nested PCR products. The overall prevalence of G. duodenalis in cattle was 12.2% (22/180), the infection being more prevalent in younger calves than in adult cattle. Zoonotic G. duodenalis Assemblage A1 was identified in both calves and workers although the most prevalent genotype detected in cattle was a novel Assemblage E subgenotype. These findings clearly suggest that there is a potential risk of zoonotic transmission of G. duodenalis infections between cattle and humans on dairy farms in India.     

Prevalence of Salmonella in dairy herds in Alberta

Fifty dairy herds in Alberta were tested for the presence of Salmonella. Four (8%) dairy herds had at least 1 cow shedding Salmonella. Different isolates were identified by serotyping, phage typing, and antibiotic resistance patterns. Pulsed-field gel electrophoresis patterns were determined for unique isolates.     

Prevalence of staphylococcal species in four dairy herds

The prevalence of staphylococcal species isolated from bovine mammary glands was determined in four dairy herds. Staphylococcus aureus and S hyicus were the predominant organisms isolated from cows in a herd with a bulk milk somatic cell count (SCC) greater than 900 X 10(3). One herd with a bulk milk SCC of 565 X 10(3) had a high incidence of S aureus while the predominant coagulase-negative species were S epidermidis and S hyicus. S hyicus predominated in two herds with bulk milk SCC less than 200 X 10(3); prevalence of S aureus was low. The impact of herd management practices such as post-milking teat antisepsis on distribution of staphylococcal species is discussed.     

Prevalence of BVDV infection in Greek dairy herds

Thirty-nine Greek dairy herds, totalling 6333 cattle, enrolled in a voluntary bovine viral diarrhoea virus (BVDV) eradication programme based on the identification and removal of persistently infected (PI) animals. The aim of this study was to estimate the prevalences of BVD antigen-positive and PI animals, and investigate the significance of the associations between the prevalence estimates and herd size. Initially, all animals were bled and examined for BVDV, using an antigen ELISA. A second sample was collected from the positive animals, after a period of at least three weeks. Animals retested positive were classified as PI. Antigen positive and PI animals were detected in all herds. The respective mean prevalences, adjusted for the test's accuracy and the herd-clustering effect, were 14% (95%CI: 11-18%) and 1.3% (0.8-1.8%), respectively. Herd size was not associated with the prevalence of antigen-positive or PI animals.     

Prevalence and intensity of infection of Cryptosporidium spp. and Giardia duodenalis in dairy cattle in Galicia (NW Spain)

Faecal samples were collected from 734 cattle selected at random from 60 dairy farms in Galicia (NW Spain). The animals studied were classified into 12 age groups: <1 month (53); 1-5 months (30); 6-11 months (31); 12-16 months (72); 17-20 months (64); 21-24 months (96); 3 years (94); 4 years (74); 5 years (67); 6 years (67); 7-8 years (63) and 9-13 years (23). Oocysts of Cryptosporidium spp. were identified in 104 animals (14.2%) distributed throughout all of the age groups and from 40 different farms (66.7%). The percentage of cattle infected ranged between 58.5% in calves <1 month and 7.9% in 7 to 8-year-old cows, i.e. the percentage of infection decreased significantly (P < 0.05) with increasing age. The intensity of infection in animals older than 1 month ranged between 10 and 5924 oocysts/g of faeces and there were no significant differences between the different groups. Cysts of Giardia duodenalis were identified in 221 animals (30.1%) from 56 farms (93.3%). The parasite was detected in all age groups, at rates of infection ranging between 21.8% (9-13 years) and 56.7% (1-5 months), although these differences were not statistically significant. The intensity of infection ranged between 7 and 15 412 cysts/g of faeces, with the number of cysts shed being significantly higher (P < 0.05) in calves <1 month than in calves aged 1-5 months. Significant associations between parasitisation by Cryptosporidium spp. or G. duodenalis and the consistency of the faeces were only found in calves aged <1 month and 1-5 months. Concurrent infections were more prevalent in the groups of calves of 1-5 months (23.3%) and 6-11 months (25.8%).     

Prevalence and genotypes of Giardia duodenalis in dairy and beef cattle in farms around Charlottetown, Prince Edward Island, Canada

Prevalence of Giardia duodenalis in dairy and beef cattle on farms around Charlottetown, Prince Edward Island (Canada) was determined by analyzing feces using direct immunofluorescence antibody microscopy. Genotypes were determined by 16S-rRNA sequencing. Fecal samples (n = 892) were collected from adult cattle in dairy tie-stall, dairy free-stall, and beef herds (10 herds each), and from calves (n = 183) from 11 dairy farms. Prevalence rates were 38% and 51% in cows and calves, respectively. Giardia duodenalis was present in all dairy herds, in 9/10 beef herds and in calves from 10/11 herds examined. Prevalence rates were 40% and 41% for cows in tie- and free-stall herds, respectively, and 27% for beef cows. Zoonotic Assemblage A was found in 12.2% of calves concomitantly infected with Assemblage E. All successfully sequenced samples (114/128) from cows corresponded to Assemblage E. Giardia duodenalis is highly prevalent in cattle herds in Prince Edward Island and Assemblage A in calves is a potential public health concern.     

Descriptive epidemiology of culling in dairy cows from 34 herds in New York State

Data on parity, disease, farmers' stated primary reasons for culling and stage of lactation at diagnosis and at culling were used to describe patterns of culling in Holstein lactations from 34 New York herds. Of 7763 lactations, 18.7% ended in culling [death (95 cows), sales for dairy purposes (104 cows), or slaughter]. The culling rates for specific reasons were: dairy purposes 1.3%, low production 3.8%, reproduction 4.8%, udder problems 4.0%, feet and legs 1.2%, old age 0.3%, accidents 0.3% and miscellaneous reasons 2.9%. Culling increased with parity (at least through Lactation 6) and primarily was due to production, reproduction and udder problems. Culling for these 3 reasons peaked immediately after calving, again between 151 and 240 days post-partum (poor milk production) and at >240 days post-partum (poor reproductive performance). First-lactation cows sold for dairy use tended to be sold in early lactation. Death in older cows usually occurred early in lactation and was due to udder problems or to miscellaneous causes.

Compared to lactations without the disease, lactations with a diagnosis of downer cow, clinical mastitis or treat problems were 3.5, 2.0 and 2.7 times more likely to end in culling, respectively. Among cows diagnosed with a disease and culled, many were culled the same day or within 30 days of the diagnosis. Such “immediate” culling upon diagnosis was especially typical of cows with milk fever, downer cow syndrome, left displaced abomasum, teat problems, and foot and leg problems (and for some cases of clinical mastitis), implying that these disorders led to “forced” culling, which was particularly costly to the farmers.     

Prevalence of Coxiella burnetii antibodies in Danish dairy herds

During recent years in Denmark higher rates of antibodies to Coxiella burnetii have been detected in animals and humans than previously reported. A study based on bulk tank milk samples from 100 randomly selected dairy herds was performed to estimate the prevalence and geographical distribution of antibody positive dairy herds. Using the CHEKIT Q-Fever Antibody ELISA Test Kit (IDEXX), the study demonstrated a prevalence of 59% antibody positive herds, 11% antibody intermediate herds and 30% antibody negative herds based on the instructions provided by the manufacturer. The geographical distribution does not indicate a relationship between the regional density of dairy farms and the prevalence of antibody positive dairy farms. The result supports the hypothesis of an increase in the prevalence of positive dairy herds compared to previous years.