Peroxidase-antiperoxidase labelling of Leishmania amastigotes in formalin fixed and paraffin embedded tissue sections.

A modified PAP-Method (peroxidase-antiperoxidase) is established for laboratory use to demonstrate Leishmania spp. amastigotes in paraffin sections of experimentally and naturally infected dogs. The results demonstrate that the PAP technique is a valuable tool to detect Leishmania amastigotes in tissue sections with low parasite density.     

Immunohistochemical detection of intermediate filament proteins in formalin fixed normal and neoplastic canine tissues.

Normal and well differentiated neoplastic canine tissues were immunohistochemically stained for keratin, vimentin and desmin intermediate filament proteins using commercially available monoclonal antibodies. Keratin was detected in 56 of 57 carcinomas, vimentin in 59 of 62 sarcomas and desmin in three of four muscle cell tumors. Most normal and neoplastic tissues expressed only one type of intermediate filament; exceptions were one hemangiosarcoma and one pulmonary carcinoma in which there was coexpression of vimentin and keratin proteins. Since immunohistochemical detection of intermediate filaments has tissue-specific distribution in the majority of well differentiated canine neoplasms, these stains may be useful in the differential diagnosis of anaplastic canine tumors. However, the monoclonal antibodies to cytokeratin which were tested in this study failed to detect intermediate filaments in liver, pancreas and salivary glands which suggests that these antibodies may also be unable to detect epithelial tumors derived from these tissues. In addition, in nine neoplasms, the normal tissues adjacent to neoplastic cells failed to stain for the intermediate filament normally expressed. When this occurs, evaluation of intermediate filament expression is invalid for the determination of tissue of origin of the neoplastic cells.     

Molecular analysis of bovine leukemia virus in early epidemic phase in Japan using archived formalin fixed paraffin embedded histopathological specimens

Bovine leukemia virus (BLV) is an important pathogen associated with enzootic bovine leukosis. In this study, we performed PCR and sequencing analysis to characterize BLVgp51 sequences from formalin-fixed paraffin-embedded (FFPE) specimens made from 1974 to 2000 and successfully obtained BLV proviral genome sequences from 94% of the analyzed samples. Furthermore, from these samples, we reconstructed eight full-length and nearly full-length BLVgp51 sequences. These sequences were classified as BLV genotype 1, implying that genotype1 has already been circulating in Japan since the 1970s. In our results, the proviral DNA was detected in the 1970s, 1980s, and 1990s in the same manner, indicating that the detection of BLV proviral genome depends on storage conditions rather than storage period. The sequences obtained in this study provide direct insights into BLV sequences before 2000, which serves as a good calibrator for inferring ancient BLV diversity.     

Demonstration of bovine virus diarrhoea virus antigen in formalin fixed, paraffin embedded tissue using a streptavidin/biotin technique

The detection of bovine virus diarrhoea virus (BVDV) antigen in sections from formalin fixed, paraffin embedded tissue is described. Pre-digestion of the sections with 0.02 per cent protease XIV for 18 hours at 4 degrees C is necessary to unmask formalin fixed antigen. A hyperimmune antiserum prepared in a pig, using a combination of BVDV and hog cholera virus inoculations, linked to a biotinylated anti-pig/streptavidin peroxidase detection system demonstrated antigen in a wide range of tissues from cases of mucosal disease and persistently viraemic animals. The inclusion of a monoclonal anti-pig immunoglobulin linked to a biotinylated anti-mouse/streptavidin peroxidase detection system greatly reduced non-specific staining.     

Immunohistochemical detection of SWC3, CD2, CD3, CD4 and CD8 antigens in paraformaldehyde fixed and paraffin embedded porcine lymphoid tissue

Identification of the different cell types of the immune system is important for in situ studies on the pathogenesis of infectious diseases in various animals, including the pig. Unfortunately, many monoclonal anti-leukocyte antibodies are only useful for staining frozen tissue sections with inherent poor tissue morphology, and are not readily adapted to formaldehyde fixed and paraffin embedded tissue with well preserved morphology. Seven well characterised monoclonal antibodies against porcine leukocyte antigens were tested on neutral buffered paraformaldehyde fixed and paraffin embedded porcine tissue sections using the highly sensitive tyramide signal amplification system. Combining this method with different antigen retrieval techniques enabled us to detect CD2, CD3, CD4, CD8 and SWC3 antigen expressing cells in porcine lymphoid tissue. Thus, we describe herein methods for the detection of several major cell types of the porcine immune system in fixed tissue with optimal preservation of histological details.     

Investigation of Cryptosporidium spp. and Giardia spp. in a Public Water‐Treatment System

The objective of this study was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in a public water‐treatment system and to relate the results to physical, chemical, bacteriological and climate parameters. From March to September 2006, 30 samples, 15 of raw water and 15 of treated water, were examined by membrane filtration and direct immunofluorescence (Merifluor). For each sample, a volume of 1000 l was collected. Of the raw‐water samples, 26.6% were positive for Cryptosporidium (mean concentration of 0.15 oocysts/l), and 6.66% were positive for Giardia (concentration of 0.2 cysts/l); 13.33% of the samples were positive for both (mean concentrations of 0.06 oocysts/l and 0.026 cysts/l respectively). All the samples of treated water were negative. There was no correlation (P < 0.05) between the presence of protozoans in the raw water and the parameters measured. The finding of Giardia and Cryptosporidium in raw water indicates that the water sources are contaminated. Considering that giardiasis is prevalent in the population and that Cryptosporidium has recognized zoonotic potential, long‐term monitoring at critical points of the system is necessary to guarantee that the water will not be a vehicle for transmission of these protozoans.     

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.     

Survey of Cryptosporidium spp. and Giardia spp. infections in various animals at a zoo in Japan.

A total of 284 fecal samples of 89 species (43 mammalian species and 46 avian species) were examined for Cryptosporidium oocysts and Giardia cysts from 1999 to 2002. Each sample was collected at the zoo located at Osaka in Japan and examined by microscopy after performing the sucrose flotation method and by two immunofluorescent assay kits for detection of Cryptosporidium oocysts and Giardia cysts. Cryptosporidium spp. was found only in a raccoon dog (Nyctereutes procyonoides), and Giardia spp. was detected in a mandarin duck (Aix galericulata) and two ruddy shelducks (Tadorna ferruginea). In this study, the prevalences of these parasites were found to be low. However, these results suggested that the infected animals could serve as a source of contamination for surface water. This is the first report about the survey of Cryptosporidium spp. and Giardia spp. at a zoo in Japan.     

Immunohistochemical detection of avian pneumovirus in formalin-fixed tissues.

An immunohistochemical staining technique (IHC) was developed to detect avian pneumovirus (APV) antigen in formalin-fixed, paraffin-embedded tissue sections using streptavidin-biotin immunoperoxidase staining. Samples of nasal turbinates and infraorbital sinuses were collected from 4-week-old poults experimentally inoculated with APV and from older turkeys infected during naturally occurring outbreaks of avian pneumovirus. Tissue was fixed in 10% buffered neutral formalin, embedded in paraffin, sectioned and stained. Inflammatory changes were observed microscopically in the mucosa and submucosa of the nasal turbinates and infraorbital sinuses of both experimentally inoculated poults and naturally infected birds. Viral antigen was detected by IHC in the ciliated epithelial cells of nasal turbinates and infraorbital sinuses.     

Prevalence and genetic characterization of Giardia spp. and Cryptosporidium spp. in dogs in Iqaluit,Nunavut, Canada

There are few epidemiologic studies on the role of dogs in zoonotic parasitic transmission in the Circumpolar North. The objectives of this study were to: (a) estimate the faecal prevalence of Giardia spp. and Cryptosporidium spp. in dogs; (b) investigate potential associations between the type of dog population and the faecal presence of Giardia spp. and Cryptosporidium spp.; and (c) describe the molecular characteristics of Giardia spp. and Cryptosporidium spp. in dogs in Iqaluit, Nunavut. We conducted two cross‐sectional studies in July and September 2016. In July, the team collected daily faecal samples for 3 days from each of 20 sled dogs. In September, the team collected three faecal samples from each of 59 sled dogs, 111 samples from shelter dogs and 104 from community dogs. We analysed faecal samples for the presence of Giardia spp. and Cryptosporidium spp. using rapid immunoassay and flotation techniques. Polymerase chain reaction (PCR) and sequencing of target genes were performed on positive faecal samples. Overall, the faecal prevalence of at least one of the target parasites, when one faecal sample was chosen at random for all dogs, was 8.16% (CI: 5.52–11.92), and for Giardia spp. and Cryptosporidium spp., prevalence was 4.42% (CI: 2.58–7.49) and 6.12% (CI: 3.88–9.53), respectively. The odds of faecal Giardia spp. in sled dogs were significantly higher than those in shelter and community dogs (OR 10.19 [CI: 1.16–89.35]). Sequence analysis revealed that 6 faecal samples were Giardia intestinalis, zoonotic assemblage B (n = 2) and species‐specific assemblages D (n = 3) and E (n = 1), and five faecal samples were Cryptosporidium canis. Giardia intestinalis is zoonotic; however, Cryptosporidium canis is rare in humans and, when present, usually occurs in immunosuppressed individuals. Dogs may be a potential source of zoonotic Giardia intestinalis assemblage B infections in residents in Iqaluit, Nunavut, Canada; however, the direction of transmission is unclear.     

Prevalence of Microsporidia, Cryptosporidium spp., and Giardia spp. in beavers (Castor canadensis) in Massachusetts.

Feces from 62 beavers (Castor canadensis) in Massachusetts were examined by fluorescence microscopy (IFA) and polymerase chain reaction (PCR) for Microsporidia species, Cryptosporidium spp., and Giardia spp. between January 2002 and December 2004. PCR-positive specimens were further examined by gene sequencing. Protist parasites were detected in 6.4% of the beavers. All were subadults and kits. Microsporidia species were not detected. Giardia spp. was detected by IFA from four beavers; Cryptosporidium spp. was also detected by IFA from two of these beavers. However, gene sequence data for the ssrRNA gene from these two Cryptosporidium spp.-positive beavers were inconclusive in identifying the species. Nucleotide sequences of the TPI, ssrRNA, and beta-giardin genes for Giardia spp. (deposited in GenBank) indicated that the four beavers were excreting Giardia duodenalis Assemblage B, the zoonotic genotype representing a potential source of waterborne Giardia spp. cysts.     

Immunohistochemical detection of canine adenovirus in paraffin sections of liver

An avidin-biotin immunoperoxidase procedure was optimized for detection of canine adenoviral antigens in formalin-fixed, paraffin-embedded liver. Long-term stability of viral antigen was shown by successful demonstration of virus in liver tissue preserved up to six years from dogs with infectious canine hepatitis. This immunohistochemical stain was applied to sections from livers with a wide range of inflammatory lesions. Examination of sections from 53 dogs yielded five livers with small amounts of adenovirus. An additional virus-positive liver was identified from a dog with no hepatic inflammation. Although a cause and effect relationship remains to be determined, these findings suggest a possible connection between canine adenovirus and spontaneous chronic hepatitis.     

Epidemiology of Cryptosporidium spp. and Giardia duodenalis on a dairy farm.

Prevalences of Cryptosporidium spp. and Giardia duodenalis in relation to age and season were investigated on a dairy farm in The Netherlands over the course of 1year. The whole herd was sampled five times, whereas calves younger than about 2 months were sampled every 2-3 weeks. Associations between diarrhoea and presence of one or more pathogens (Cryptosporidium spp., G. duodenalis, rotavirus) were investigated. Potential transmission routes of Cryptosporidium spp. were evaluated and positive samples of Cryptosporidium spp. and G. duodenalis were identified to genotype level by PCR microsatellite identification and fingerprinting. Shedding of Cryptosporidium spp. was found in all age categories but peaked in calves 1-3 weeks old (39.1%). Herd prevalence of shedding for Cryptosporidium spp. varied from 2.4% in June to 22.2% in December. Shedding of G. duodenalis was found in all age categories but peaked in animals 4-5 months old (54.5%). Herd prevalence of shedding for G. duodenalis varied from 0.8% in June to 15.5% in February. Cryptosporidium spp. and rotavirus appeared to be significantly associated with diarrhoea in calves. Microsatellite analysis showed two different subtypes (C3 and C1) of Cryptosporidium parvum calf strains. Two genotypes of G. duodenalis were found, one positive by A lineage specific PCR and thus closely related to human genotypes and one genotype, which was negative by A and B lineage specific PCR. The results indicate that cow-to-calf and indirect calf-to-calf transmission both are important routes for acquiring infection with Cryptosporidium spp.     

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.     

Giardia duodenalis and Cryptosporidium spp. in a veterinary college bovine teaching herd

In a preliminary study, we commonly identified Giardia duodenalis in adult dairy cattle from a veterinary college teaching herd. Therefore, the present study was carried out in order to better understand the potential of adult cattle to act as a source for G. duodenalis infections for students and staff at the veterinary college. Fecal samples were collected bi-weekly from this herd of adult cattle (n=30) over an 8-month period to determine the prevalence of G. duodenalis and Cryptosporidium spp. within the herd. Nested PCR followed by DNA sequencing was then performed on a subset of positive samples in order to better understand the zoonotic potential of these infections. Every cow was sampled between 11 and 18 times, depending on the date the animal joined the teaching herd. In total, 507 fecal samples were collected from 30 different cows and examined for cysts and oocysts using epifluorescence microscopy. G. duodenalis prevalence during the course of the study ranged from 37% (11/30) to 64% (18/28), with a mean of 49%. Cumulative G. duodenalis prevalence was 73% (22/30). Zoonotic G. duodenalis assemblage A genotype was identified in 43% (6/14) of the G. duodenalis-positive samples on which PCR and genetic sequencing were successfully performed. G. duodenalis assemblage E was identified in 57% (8/14) of these samples. Cryptosporidium spp. oocysts were not detected in the feces of any cows during the study period. The presence of the zoonotic G. duodenalis assemblage A in 43% of the sequenced samples indicates that there is a potential risk of infection for students and staff at this research and teaching facility, although the roles of cows as sources of giardiasis in humans remain uncertain. Furthermore, due to the large amount of feces they produce, adult cattle may serve as important sources for G. duodenalis infections in young cattle, or other animals in the facility, despite relatively low numbers of cysts excreted per gram of feces. In contrast, the results of this study indicate that this herd posed a negligible risk of transmitting Cryptosporidium parvum infections to humans.     

Prevalence and factors associated with fecal shedding of Giardia spp. in domestic cats

The prevalence of cats shedding Giardia cysts (13.6%) in the present study was found to be higher than previously reported (1% to 11%) and may reflect a higher sensitivity for the diagnostic test used. The presence of Cryptosporidium spp. oocysts, coccidial oocysts, and a clinical history of chronic (>2 weeks) gastrointestinal signs were significantly associated with the presence of Giardia spp. cysts in the feces. There were no associations between the presence of Giardia spp. cysts and type of housing, acute gastrointestinal signs, vomiting, gender, source of cat (i.e., animal shelter versus private breeder), or gastrointestinal parasites other than Cryptosporidium spp. and intestinal coccidial agents.