Gas chromatography-mass spectrometry-based metabolite profiling of Salmonella enterica serovar Typhimurium differentiates between biofilm and planktonic phenotypes.

The aim of this study was to utilize gas chromatography coupled with mass spectrometry (GC-MS) to compare and identify patterns of biochemical change between Salmonella cells grown in planktonic and biofilm phases and Salmonella biofilms of different ages. Our results showed a clear separation between planktonic and biofilm modes of growth. The majority of metabolites contributing to variance between planktonic and biofilm supernatants were identified as amino acids, including alanine, glutamic acid, glycine, and ornithine. Metabolites contributing to variance in intracellular profiles were identified as succinic acid, putrescine, pyroglutamic acid, and N-acetylglutamic acid. Principal-component analysis revealed no significant differences between the various ages of intracellular profiles, which would otherwise allow differentiation of biofilm cells on the basis of age. A shifting pattern across the score plot was illustrated when analyzing extracellular metabolites sampled from different days of biofilm growth, and amino acids were again identified as the metabolites contributing most to variance. An understanding of biofilm-specific metabolic responses to perturbations, especially antibiotics, can lead to the identification of novel drug targets and potential therapies for combating biofilm-associated diseases. We concluded that under the conditions of this study, GC-MS can be successfully applied as a high-throughput technique for "bottom-up" metabolomic biofilm research.

Comparative susceptibility of Salmonella Typhimurium biofilms of different ages to disinfectants.

There is a general consensus that with increasing age a biofilm shows increased resistance to antimicrobials. In this study the susceptibility of 3-, 5- and 7-day-old Salmonella enterica serovar Typhimurium biofilms to disinfectants was evaluated. It was hypothesized that 7-day-old biofilms would be more resistant to disinfectants compared to 3- and 5-day-old biofilms. Biofilms were formed using the MBEC™ system and treated with six chemical disinfectants for 1 and 5 min. Four disinfectants at the highest concentration available showed 100% reduction in viable cells from all ages of biofilms after exposure for 5 min, and ethanol at 70% v/v was the least effective against biofilms, followed by chlorhexidine gluconate (CG). At the recommended user concentrations, only sodium hypochlorite showed 100% reduction in viable cells from all ages of biofilms. Benzalkonium chloride and CG were the least effective against biofilms, followed by quaternary ammonium compound which only showed 100% reduction in viable cells from 5-day-old biofilms. Overall, the results from this study do not display enhanced resistance in 7-day-old biofilms compared to 3- and 5-day-old biofilms. It is concluded that under the conditions of this study, the age of biofilm did not contribute to resistance towards disinfectants. Rather, the concentration of disinfectant and an increased contact time were both shown to play a role in successful sanitization.

Prevalence and molecular characterization of Cryptosporidium spp. in dairy calves from 11 farms in Prince Edward Island, Canada.

Cryptosporidium spp. are common intestinal protozoan parasites that infect a wide range of hosts, including humans and livestock, worldwide. The objective of this study was to determine the prevalence of Cryptosporidium spp. in dairy calves in Prince Edward Island, Canada, and the potential for transmission of this parasite between dairy calves and humans. Fecal samples were collected from 183 dairy calves from 11 farms in Prince Edward Island. The prevalence of Cryptosporidium spp. infections in these animals was determined by examining for the presence of oocysts in the fecal samples, using immunofluorescence microscopy. Molecular characterization was done using a nested-PCR protocol to amplify fragments of the Cryptosporidium heat-shock protein 70 gene, followed by DNA sequencing. Ten calves (6.2%), representing 4 out of 11 farms tested, were positive for Cryptosporidium spp. DNA sequence analysis on five PCR positive samples demonstrated that Cryptosporidium parvum was the only species present in the calves tested, suggesting that there is a potential risk of zoonotic transmission between dairy calves and humans in this region.

Aminoguanidines: New leads for treatment of Giardia duodenalis infection.

Giardia duodenalis is an ubiquitous parasitic pathogen that causes significant morbidity and mortality worldwide. Failures in drug therapy are commonly due to poor patient compliance as a result of the need for repeated administration, off target drug effects and increasing parasite drug resistance. In this study the in vitro efficacy and selectivity of the aminoguanidine compound robenidine and 2 structural analogues against Giardia were determined. After 5 h exposure to each compound the IC50 was as low as 0.2 µM with corresponding MLCs as low as 2.8 µM. This is in contrast to metronidazole which required 24 h to exhibit inhibitory activity. A modified adherence assay, developed for this study, demonstrated that three of the compounds inhibited in vitro adherence of the parasite. The lead compound exhibited rapid giardicidal activity (<5hr). In addition, microscopy studies demonstrated damage to the plasma membrane of trophozoites. In conclusion, a class of aminoguanidines, represented by robenidine, has shown antigiardial activity warranting further investigation.

Comparison of flow cytometry and immunofluorescence microscopy for the detection of Giardia duodenalis in bovine fecal samples.

The performance of flow cytometry (FC) was compared with immunofluorescence microscopy (IM) for detection of Giardia duodenalis in bovine feces. Samples from 36 adult dairy cows and 208 dairy calves were collected. Flow cytometry test characteristics were calculated using continuous, ordinal, and dichotomized results. Spearman correlation coefficients comparing the results of the 2 tests were 0.47 and 0.68 for cows and calves, respectively. Using IM as indicative of presence or absence of G. duodenalis cysts in each sample, likelihood ratios of FC results with 0, 1, and > or = 2 gated events indicated that samples with 1 gated event were likely to be positive in the cows but not in the calves. Immunofluorescence microscopy detected G. duodenalis in 69.7% and 48.1% of cows and calves, respectively. When dichotomizing the FC results at a cut-off point of 1 or 2 gated events, 46.3% and 19.9% of the cow and 51.9% and 35.1% of the calf samples, respectively, were classified as G. duodenalis-positive. Relative to IM, the sensitivity in the cows was 0.59 and 0.28, respectively, and 0.76 and 0.64, respectively, in the calves. At a cut-off point of 1, 65.7% and 73.1% of the cow and calf samples, respectively, were correctly classified in FC, and at a cut-off point of 2, 49.3% and 78.4% were correctly classified in the cows and calves, respectively. Flow cytometry was less sensitive than IM. Possible reasons and research needed to improve FC for G. duodenalis detection are discussed.

Cryptosporidium parvum infection in cattle: are current perceptions accurate?

The perception that cattle are major reservoirs for Cryptosporidium parvum infections in humans and that C. parvum is a major cause of diarrhea and production loss in cattle might not reflect the whole situation. Numerous management factors influence the epidemiological and clinical picture associated with C. parvum infections in cattle. Whereas C. parvum is highly prevalent in young dairy calves and confined beef calves, it occurs rarely in calves on range and in adult cattle. In well-managed herds, clinical disease due to C. parvum is also rare. Therefore, C. parvum infections in cattle might not be as important as current perceptions would indicate.

Efficacy of vaccination in preventing giardiasis in calves.

The objective of this study was to evaluate the efficacy of a vaccine in the prevention of Giardia duodenalis infection in calves. Six 2-week old calves were vaccinated subcutaneously with a sonicated G. duodenalis trophozoite vaccine. Six 2-week old control calves received a subcutaneous injection of sterile phosphate-buffered-saline mixed with adjuvant. Injections were repeated after 28 days. Eleven days after the second injection, calves were challenged orally with 1x10(5) purified G. duodenalis cysts from a naturally infected calf. Throughout the study, fecal samples were collected at regular intervals and examined for the presence of G. duodenalis cysts. Blood samples were collected weekly until G. duodenalis challenge and bi-weekly following challenge. Calves were euthanized 14 days after challenge and G. duodenalis trophozoites within the small intestines were enumerated. Serum antibody titers were significantly higher in vaccinated compared to non-vaccinated calves. Vaccinated calves tended to excrete more G. duodenalis cysts in their feces than non-vaccinated calves. The number of trophozoites in the small intestine was not different between vaccinated and non-vaccinated calves. Changes consistent of moderate enteritis were found in the intestines of one vaccinated and one non-vaccinated calf. Despite a serological immune response following vaccination, this vaccine was not efficacious in preventing giardiasis or reducing cyst shedding in calves.

An outbreak of dictyocaulosis in lactating cows on a dairy farm.

CASE DESCRIPTION: The owner of a herd of 74 Holstein-Friesian cattle reported decreased milk production, weight loss, and coughing among lactating cows. Owner-initiated antimicrobial treatment was unsuccessful; 1 lactating cow died, and 50% of the lactating cows had clinical signs of respiratory distress, such as tachypnea and coughing. CLINICAL FINDINGS: On the basis of history, physical examination findings, and fecal examination results, affected animals were determined to have Dictyocaulus viviparus (lungworm) infestation. The disease history suggested that the herd contained cows with subclinical patent lungworm infestations; after introduction of susceptible heifers, the pastures had become heavily infested with D viviparus and clinical problems subsequently developed in both newly introduced and resident cows. TREATMENT AND OUTCOME: Affected and unaffected heifers and adult cows were treated with a pour-on formulation of eprinomectin (0.5 mg/kg [0.23 mg/lb]). One animal died, but 2 weeks after treatment, clinical signs among affected cattle were markedly improved. Ten weeks after treatment, milk production improved from 23 kg/cow/d (51 lb/cow/d) to 28 kg/cow/d (62 lb/cow/d). CLINICAL RELEVANCE: The outbreak provides additional evidence that dictyocaulosis is becoming more common among adult dairy cattle, rather than almost exclusively affecting young stock. This may be attributable to anthelmintic use and management practices on dairy farms. Combined with anecdotal reports of an increase in the incidence of dictyocaulosis among adult cattle in North America, D viviparus infestation should be included as a differential diagnosis for decreased milk production, weight loss, and coughing among adult dairy cattle.

Use of an enzyme-linked immunosorbent assay in bulk milk to estimate the prevalence of Neospora caninum on dairy farms in Prince Edward Island, Canada.

This study evaluated the use of bulk milk as a diagnostic tool for estimation of herd-level Neospora caninum exposure in Atlantic Canada; it was used to estimate the prevalence of dairy farms with a within-herd N. caninum-seroprevalence > or = 15% in Prince Edward Island (PEI). The variation over time of N. caninum antibodies in bulk milk is also reported. Skimmed bulk milk and individual serum samples were analyzed for N. caninum antibodies by using an enzyme-linked immunosorbent assay (ELISA). Bulk milk samples were collected in May 2004 (n = 235), May 2005 (n = 189), and June 2005 (n = 235). The prevalence of dairy farms with a within-herd seroprevalence > or = 15% on PEI was 6.4% in May 2004. In May and June 2005, respectively, 10.1% and 10.2% of farms had a > or = 15% within-herd seroprevalence. In 11 farms that were considered positive based on bulk milk samples, blood samples were collected from all adult cows in September 2005, in conjunction with a 4th bulk milk sample on the same day. The correlation coefficient between serology and bulk milk ELISA was 0.87. The results of this study demonstrate that the prevalence of N. caninum in dairy farms can be estimated by using a bulk milk ELISA.

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.

Neospora caninum-like oocysts observed in feces of free-ranging red foxes (Vulpes vulpes) and coyotes (Canis latrans).

The aim of this study was to examine the feces of free-ranging foxes and coyotes for the presence of Neospora caninum oocysts. Feces were collected from 271 foxes and 185 coyotes in the Canadian province of Prince Edward Island, processed by sucrose flotation, and examined by light microscopy for the presence of coccidian oocysts. In 2 fox and 2 coyote samples, oocysts morphologically and morphometrically similar to oocysts of N. caninum were observed. DNA was extracted from these samples and subjected to nested polymerase chain reaction (PCR) using primers to the N. caninum-specific Nc5 genomic sequence. Through DNA sequencing, alignment of the sequences of at least 3 clones from each isolate to sequences deposited in GenBank revealed 95-99% similarity to the Nc5 sequence of N. caninum. PCR using primers specific for Hammondia heydorni failed to yield an amplification product from these DNA samples.

Giardiasis and cryptosporidiosis in ruminants.

Although they differ considerably with respect to their biology, both Giardia duodenalis and Cryptosporidium parvum are common in ruminants, whereas Cryptosporidium andersoni is not. G. duodenalis infections are acquired during the first few months of life, tend to be chronic, and may be a production-limiting disease of ruminants. C. parvum infections remain an important cause of diarrhea in neonatal ruminants. Abomasal cryptosporidiosis, caused by C. andersoni, is an emerging disease of cattle that may affect both beef and dairy herds. This article reviews the life cycles, production impacts, treatments, controls, and zoonotic potentials of these important ruminant parasites.

Passive immunity and serological immune response in dairy calves associated with natural Giardia duodenalis infections.

In a previous study, Giardia infection patterns were studied in newborn dairy calves over a 4-month period. Chronic Giardia infections were observed in all calves with initial cyst excretion occurring at approximately 1 month of age. In the work presented here, the passive immunity and serological immune response associated with these Giardia infections were examined. Colostrum and milk samples were collected from the dams of these calves, and monthly serum samples were collected from each calf. The colostrum, milk and sera samples were analyzed by ELISA and Western blot for the presence of anti-Giardia IgG antibodies. In addition, the in vitro anti-Giardia activity of milk and colostrum was examined using a miniculture adherence assay. When examined by ELISA, mean anti-Giardia antibody titres were found to be significantly higher in colostrum compared to milk. The monthly mean serum antibody titres in the calves were not found to differ significantly at any time point during the study. Western blot analysis revealed that colostrum from the dams reacted strongly with many different Giardia antigens between 205 and 7.5kDa, while milk reacted with few antigens in the same size range. Sera collected from the calves when 30 and 60 days of age reacted with few Giardia antigens, but as the calves aged, IgG antibodies in their sera began to react with antigens of 21, 50, 65, 73 and 79kDa. The miniculture adherence assay demonstrated that colostrum had significantly more anti-Giardia activity in vitro compared to milk. These results suggest that the calves in this dairy did not mount a significant humoral immune response against Giardia following infection. However, colostrum contained a high level of anti-Giardia antibodies and exhibited anti-Giardia activity in vitro. Therefore, colostrum may have the potential to provide initial protection against Giardia infections in calves, but the lack of a strong, specific humoral immune response by these calves could account for the high prevalence and chronic duration of the infections.