A four year longitudinal sero-epidemiology study of <Emphasis Type="Italic">Neospora caninum</Emphasis>in adult cattle from 114 cattle herds in south west England: Associations with age,herd and dam-offspring pairs

Background

Neosporosis caused by the protozoan parasite Neospora caninum, is an economically important cause of abortion, stillbirth, low milk yield, reduced weight gain and premature culling in cattle. Consequently, a seroepidemiological study of N. caninum antibodies was conducted in England with 29,782 samples of blood taken from 15,736 cattle from 114 herds visited on three occasions at yearly intervals. Herds were categorised into lower (< 10%) and higher (≥ 10%) median herd seroprevalence. Hierarchical models were run to investigate associations between the sample to positive (S/P) ratio and herd and cattle factors.

Results

Ninety-four percent of herds had at least one seropositive cow; 12.9% of adult cattle had at least one seropositive test. Approximately 90% of herds were seropositive at all visits; 9 herds (8%) changed serological status between visits. The median N. caninum seroprevalence in positive herds was 10% (range 0.4% to 58.8%). There was a positive association between the serostatus of offspring and dams that were ever seropositive. In the hierarchical model of low seroprevalence herds there was no significant association between S/P ratio and cattle age. There was a significantly lower S/P ratio in cattle in herds that were totally restocked after the foot-and-mouth epidemic of 2001 compared with those from continuously stocked herds and cattle purchased into these herds had a higher S/P ratio than homebred cattle. In the model of high seroprevalence herds the S/P ratio increased with cattle age, but was not associated with restocking or cattle origin.

Conclusion

There were no strong temporal changes in herd seroprevalence of N. caninum but 90% of herds had some seropositive cattle over this time period. Vertical transmission from seropositive dams appeared to occur in all herds. In herds with a high seroprevalence the increasing S/P ratio in 2–4 year old cattle is suggestive of exposure to N. caninum: horizontal transmission between adult cattle, infection from a local source or recrudescence and abortions. Between-herd movements of infected cattle enhance the spread of N. caninum, particularly into low seroprevalence herds. Some restocked herds had little exposure to N. caninum, while in others infection had spread in the time since restocking.

     

Questionnaire identifying management practices surrounding calving on spring-calving dairy farms and their associations with herd size and herd expansion

Healthy calves are fundamental to any profitable dairy enterprise. Research to-date, has focused on year-round calving systems which experience many different challenges compared to spring-calving systems. The objective of the present study was to determine the on-farm dry cow, calving, and colostrum management practices of spring-calving dairy production systems, and quantify their associations with herd size and herd expansion status (i.e. expanding or not expanding). Information on these management practices was available from a survey of 262 Irish spring-calving dairy farmers, representative of the Irish national population. Herd expansion in the 2 years before, and the year that the survey was conducted was not associated with any of the management practices investigated. Fifty-three percent of respondents had an average calving season length of 10 to14 weeks with 35% of herds having a longer calving season. Previous research in cattle has documented that both colostrum source and feeding management are associated with the transmission of infectious disease from cow to calf. In the present study 60% of respondents fed calves colostrum from their own dam; however, 66% of those respondents allowed the calf to suckle the dam, 23% of survey respondents fed calves pooled colostrum. Larger herds were more likely (P<0.01) to use pooled colostrum supplies, while smaller herds were more likely (P<0.05) to allow the calf to suckle the dam. The majority (86%) of respondents had stored supplies of colostrum; average-sized herds had the greatest likelihood of storing colostrum (P<0.05), compared to other herd sizes; larger sized herds had a lesser likelihood (P<0.05) of storing colostrum in a freezer, compared to other herd sizes. Although freezing colostrum was the most common method used to store colostrum (54% of respondents), 17% of respondents stored colostrum at room temperature, 29% of which stored it at room temperature for greater than 4 days. The results from the present study indicate that a particular focus needs to be placed on calving and colostrum management because this study has highlighted a number of areas which are below international standards, and may have repercussions for calf health. Furthermore, management practices on larger farms could be improved and, as these represent the future of dairy farming, a focus needs to be placed on them. Expanding herds are not a particular concern as herd expansion, independent of herd size, does not seem to be associated with calving and colostrum management practices on Irish spring-calving dairy herds.     

Management practices associated with reproductive performance in Holstein cows on large commercial dairy farms

As a result of the increase in herd size and the intensification of production, the complexity of reproductive management has been growing in dairy herds. The aim of our study was to examine the associations of management practices and reproductive performance in Holstein cows on large commercial dairy farms. Management practices applied to cows were surveyed between 22 May and 6 November 2015 in 34 large Holstein-Friesian dairy herds in Hungary. Individual data of 23 784 cows that calved between 1 January 2014 and 31 December 2014 in the surveyed herds were gathered. Associations between the management practices and the reproductive parameters were analyzed by mixed effects models. Regarding heat abatement we found that ventilation with sprinklers was associated with the shortest breeding interval (P<0.01), the shortest calving to conception interval (P<0.01), and the highest odds of being pregnant by 200 days in milk (P<0.01). Solely ventilation showed similar (P>0.05) results to lack of heat stress protection. It was also revealed, that lack of a well-established voluntary waiting period (VWP) or a VWP shorter than 50 days was associated with less days to first service (P<0.01), shorter breeding interval (P<0.01) and calving to conception interval (P<0.05), as well as higher odds of carrying a calf by 200 days in milk (P<0.01) compared with those using a VWP of at least 50 days. Those farms that applied transrectal ultrasonography were more likely to use ventilation with sprinklers (P<0.05), hormonal synchronization (P<0.01) and to perform early pregnancy diagnosis followed by pregnancy recheck (P<0.05). The application of transrectal ultrasonography with one of the aforementioned practices was associated with reduced days to first service (P<0.05), shorter breeding interval (P<0.05) and higher odds of pregnancy at 200 days in milk (P<0.05). Our study highlights the management practices most closely related to improved reproductive performance, which are, therefore, suggested to be applied on dairy farms, considering the local circumstances of the individual farms.     

Development of Two Murine Antibodies against Neospora caninum Using Phage Display Technology and Application on the Detection of N. caninum

Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1) and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA) with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection.     

Fecal and Molecular Survey of Neospora caninum in Farm and Household Dogs in Mashhad Area, Khorasan Province, Iran

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dog is the definitive host for N. caninum and can infect dairy cattle. The aim of this study is to determine the prevalence of Neospora oocysts in feces of dogs from dairy farms. A total of 174 fecal samples was collected from 89 farm dogs and 85 household dogs during 2006 and 2008. Fecal samples of dogs were microscopically examined for detecting Hammondia Neospora-like oocysts (HNLO) by Mini Parasep®SF fecal parasite concentrator. HNLO were microscopically detected in 4 fecal samples (2.2%). The fecal samples with HNLO were examined by N. caninum-specific PCR. Two of the samples were positive for N. caninum. The 2 positive fecal samples were selected for inoculation to calves. Two inoculated calves were seronegative by ELISA for 4 months post-infection. This is the first report of finding N. caninum DNA in feces of farm dogs in Mashhad area, Iran.     

Effect of cow replacement strategy on cow and calf performance in the beef herd

Two contrasting replacement strategies are used by Irish beef farmers to select replacement females – animals sourced from within the suckler beef herd and sourced from the dairy herd. The objective of this study was to investigate the effect of replacement strategy (i.e. beef v. beef×dairy (BDX)) on cow and calf performance using data from the national beef database across a range of beef and dairy breeds. The association between replacement strategy and calving difficulty score, calving interval, weaning weight, weaning price and all carcass traits was investigated using a mixed model. The effect of replacement strategy on cow survival, calving dystocia and calf perinatal mortality was quantified using logistic regression. Beef cows were older (10.92 days; P<0.001) at their first calving, but were 1.15 times (P<0.01) more likely to survive to a subsequent lactation compared with BDX cows. Calving interval was 1.53 days shorter (P<0.001) for BDX compared with beef cows. Greater calving difficulty and calving dystocia was associated with beef cows (P<0.001) relative to BDX. However, BDX were 1.36 times (P<0.001) more likely to have a dead calf at birth relative to beef cows. Calves weaned from BDX were heavier (18.49 kg; P<0.001) at weaning, reached slaughter 12.8 days earlier (P<0.001), had 7.99 kg heavier carcass (P<0.001) and a greater fat score (P<0.001) compared with the progeny of beef cows. Beef cow progeny had a superior conformation score (0.5; P<0.001) and achieved a greater price per kilogram (P<0.001) compared with progeny from BDX. Beef cull cows had a heavier carcass (5.58 kg), superior carcass conformation, greater carcass price per kilogram and greater overall carcass value (P<0.001) than BDX. Results from this study show that replacement strategy is of fundamental importance depending on the type of system implemented by farmers and consideration must be given to the traits of importance within the context of the individual production system.     

Survival of Salmonella enterica Serovar Newport in Manure and Manure-Amended Soils

Salmonella enterica serovar Newport has undergone a rapid epidemic spread in dairy cattle. This provides an efficient mechanism for pathogen amplification and dissemination into the environment through manure spreading on agricultural land. The objective of this study was to determine the survival characteristics of Salmonella serovar Newport in manure and manure-amended soils where the pathogen may be amplified. A multidrug-resistant (MDR) Salmonella serovar Newport strain and a drug-susceptible (DS) strain, both bovine isolates, were inoculated into dairy manure that was incubated under constant temperature and moisture conditions alone or after being mixed with sterilized or nonsterilized soil. Salmonella serovar Newport concentrations increased by up to 400% in the first 1 to 3 days following inoculation, and a trend of steady decline followed. With manure treatment, a sharp decline in cell concentration occurred after day 35, possibly due to microbial antagonism. For all treatments, decreases in Salmonella serovar Newport concentrations over time fit a first-order kinetic model. Log reduction time was 14 to 32 days for 1 log₁₀, 28 to 64 days for 2 log₁₀, and 42 to 96 days for 3 log₁₀ declines in the organisms' populations from initially inoculated concentrations. Most-probable-number monitoring data indicated that the organisms persisted for 184, 332, and 405 days in manure, manure-amended nonsterilized soil, and manure-amended sterilized soil, respectively. The MDR strain and the DS strain had similar survival patterns.     

Serological evidence of Toxoplasma gondii and Neospora caninum infection in black-boned sheep and goats in southwest China

Toxoplasma gondii and Neospora caninum are two closely related protozoan parasites which can cause abortion and significant economic losses in sheep and goats. However, it is yet to know whether black-bone sheep and goats are infected with T. gondii and N. caninum in China. In the present investigation, the seroprevalence and risk factors of T. gondii and N. caninum infections in black-boned sheep and goats were investigated in Yunnan Province, subtropical southwest China between July and August of 2017. A total of 481 serum samples were tested for T. gondii antibodies using the Modified Agglutination Test (MAT), and 468 serum samples were examined for N. caninum antibodies by indirect Enzyme-Linked Immunosorbent Assay (iELISA). The overall seroprevalence of T. gondii in black-boned sheep and goats was 36.80% (177/481, 95% CI 32.49–41.11), and 40 out of 468 serum samples were N. caninum-seropositive (8.55%, 95% CI 6.02–11.08). There was significant difference in the seroprevalence of T. gondii infection in different regions (χ² = 19.869, df = 2, P<0.01). As for the seroprevalence of N. caninum infection, region (χ² = 8.558, df = 2, P<0.05), age (χ² = 16.631, df = 3, P < 0.01), gender (χ² = 11.219, df = 1, P < 0.01) and species (χ² = 8.673, df = 1, P < 0.01) were the risk factors. In addition, the seroprevalence of coinfection of T. gondii and N. caninum in black-boned sheep and goats was 3.63% (17/468, 95% CI 1.94–5.32). To our knowledge, this is the first report of T. gondii and N. caninum seroprevalence in black-boned sheep and goats in China, which provided base-line data for the execution of control strategies and measures against T. gondii and N. caninum infection in black-boned sheep and goats.     

Australian dingoes are definitive hosts of Neospora caninum

To provide objective data on the potential role of dingoes (Canis lupus dingo) in the life cycle of Neospora caninum in Australia, the production of N. caninum oocysts by experimentally infected canids was investigated. Three dingo pups raised in captivity and three domestic dogs were fed tissue from calves infected with an Australian isolate of N. caninum, Nc-Nowra. Oocysts of N. caninum, confirmed by species-specific PCR, were shed in low numbers by one dingo pup at 12-14 days p.i. The remaining animals did not shed oocysts. Furthermore, the blood from two out of three dingoes tested positive for DNA of N. caninum using PCR tests at 14 and 28 days p.i. Oocyst shedding from the intestinal tract of a dingo demonstrates that dingoes are definitive hosts of N. caninum and horizontal transmission of N. caninum from dingoes to farm animals and wildlife may occur in Australia.     

Internal teat sealants alone or in combination with antibiotics at dry-off – the effect on udder health in dairy cows in five commercial herds

In the dairy industry, the dry period has been identified as an area for potential reduction in antibiotic use, as part of a one health approach to preserve antibiotic medicines for human health. The objective of this study was to assess the impact of dry cow treatment on somatic cell count (SCC), intramammary infection (IMI) and milk yield on five commercial Irish dairy herds. A total of 842 cows across five spring calving dairy herds with a monthly bulk tank SCC of < 200 000 cells/mL were recruited for this study. At dry-off, cows which had not exceeded 200 000 cells/mL in the previous lactation were assigned one of two dry-off treatments: internal teat seal (ITS) alone (Lo_TS) or antibiotic plus ITS (Lo_AB + TS). Cows which exceeded 200 000 cells/mL in the previous lactation were treated with antibiotic plus ITS and included in the analysis as a separate group (Hi_AB + TS). Test-day SCC and lactation milk yield records were provided by the herd owners. Quarter milk samples were collected at dry-off, after calving and at mid-lactation for bacteriological culture and quarter SCC analysis. Cow level SCC was available for 789 cows and was log-transformed for the purpose of analysis. Overall, the log SCC of the cows in the Lo_TS group was significantly higher than the cows in Lo_AB + TS group and not statistically different to the cows in the Hi_AB + TS group in the subsequent lactation. However, the response to treatment differed according to the herd studied; the log SCC of the cows in the Lo_TS group in Herds 3, 4 and 5 was not statistically different to the cows in Lo_AB + TS group, whereas in the other two herds, the log SCC was significantly higher in the Lo_TS when compared to the Lo_AB + TS group. There was a significant interaction between dry-off group and herds on SCC and odds of infection in the subsequent lactation. The results of this study suggest that the herd prevalence of IMI may be useful in decision-making regarding the treatment of cows with ITS alone at dry-off to mitigate its impact on udder health.     

Neospora caninum in birds: A review

Neospora caninum is an obligate intracellular protozoan parasite that infects domestic and wild animals. Canids are considered to be definitive hosts since they may shed oocysts into the environment through their feces. The disease is recognized as one of the major causes of bovine abortion worldwide, leading to important economic losses in the dairy and beef cattle industries. Previous studies have reported N. caninum infection in different species of birds; infection in birds has been associated with increased seroprevalence and reproductive problems in dairy cattle. Although the role of birds in the epidemiological cycle of neosporosis is unknown, birds are exposed to infection because they feed on the ground and could thus contribute to parasite dissemination. This review is focused on the current state of knowledge of neosporosis in birds.     

Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells

Background

Neosporosis is an infectious disease primarily of cattle and dogs, caused by intracellular parasite, Neospora caninum. Neosporosis appears to be a major cause of abortion in dairy cattle worldwide and causes to huge economic loss to dairy industry.

Results

Recombinant surface associated antigen 1 (NcSAG1), NcSAG1 related sequence 2 (NcSRS2) and the dense granule antigen 2 (NcGRA2) of N. caninum were expressed either in silkworm or in Escherichia coli and purified. The purified recombinant proteins bound to the N. caninum-specific antibodies in serum samples from infected cattle as revealed by an enzyme-linked immunosorbent assay (ELISA). By co-immobilizing these recombinant proteins, a novel indirect ELISA was developed for detection of neosporosis. With the use of 32 serum samples, comprising 12 positive serum samples and 20 negative serum samples, the sensitivity and specificity of the assay were found to be 91.7 and 100%, respectively. Seventy-two serum samples from dairy farms were also tested and one was diagnosed with neosporasis with both this method and a commercial assay.

Conclusions

A diagnostic method employing recombinant proteins of N. caninum was developed. The method showed high sensitivity and specificity. Diagnostic test with field serum samples suggested its applicability to the practical diagnosis of neosporosis.     

Factors associated with the concentration of immunoglobulin G in the colostrum of dairy cows

Transfer of sufficient immunoglobulin G (IgG) to the neonatal calf via colostrum is vital to provide the calf with immunological protection and resistance against disease. The objective of the present study was to determine the factors associated with both colostral IgG concentration and colostral weight in Irish dairy cows. Fresh colostrum samples were collected from 704 dairy cows of varying breed and parity from four Irish research farms between January and December 2011; colostral weight was recorded and the IgG concentration was determined using an ELISA method. The mean IgG concentration in the colostrum was 112 g/l (s.d. = 51 g/l) and ranged from 13 to 256 g/l. In total, 96% of the samples in this study contained >50 g/l IgG, which is considered to be indicative of high-quality colostrum. Mean colostral weight was 6.7 kg (s.d. = 3.6 kg) with a range of 0.1 to 24 kg. Factors associated with both colostral IgG concentration and colostral weight were determined using a fixed effects multiple regression model. Parity, time interval from calving to next milking, month of calving, colostral weight and herd were all independently associated with IgG concentration. IgG concentration decreased (P < 0.01) by 1.7 (s.e. = 0.6) g/l per kg increase in the colostral weight. Older parity cows, cows that had a shorter time interval from calving to milking, and cows that calved earlier in spring or in the autumn produced colostrum with higher IgG concentration. Parity (P < 0.001), time interval from calving to milking (P < 0.01), weight of the calf at birth (P < 0.05), colostral IgG concentration (P < 0.01) and herd were all independently associated with colostral weight at the first milking. Younger parity cows, cows milked earlier post-calving, and cows with lighter calves produced less colostrum. In general, colostrum quality of cows in this study was higher than in many previous studies; possible reasons include use of a relatively low-yielding cow type that produces low weight of colostrum, short calving to colostrum collection interval and grass-based nutritional management. The results of this study indicate that colostral IgG concentration can be maximised by reducing the time interval between calving and collection of colostrum.     

Prevalence and Fimbrial Genotype Distribution of Poultry Salmonella Isolates in China (2006 to 2012)

In this study, a total of 323 Salmonella enterica strains were isolated from 3,566 rectal swab samples of 51 poultry farms in seven regions of 12 provinces of China between 2006 and 2012. The prevalences of Salmonella sp. carriage were 12.4% in geese (66 positive/533 samples), 10.4% in turkeys (32/309), 9.8% in chickens (167/1,706), 6.8% in ducks (41/601), and 4.1% in pigeons (17/417), respectively. These isolates belonged to 20 serovars, in which the most frequent serovars were S. enterica serovar Gallinarum biovar Pullorum (herein, S. Pullorum) (55 isolates, 17.0%), S. enterica serovar Typhimurium (50 isolates, 15.5%), and S. enterica serovar Enteritidis (39 isolates, 12.1%). Overall, S. Typhimurium was the most commonly detected serovar; among the individual species, S. Pullorum was most commonly isolated from chickens, S. Enteritidis was most common in ducks, S. Typhimurium was most common in geese and pigeons, and S. enterica serovar Saintpaul was most common in turkeys. PCR determination of 20 fimbrial genes demonstrated the presence of bcfD, csgA, fimA, stdB, and sthE genes and the absence of staA and stgA genes in these isolates, and other loci were variably distributed, with frequency values ranging from 11.8 to 99.1%. These 323 Salmonella isolates were subdivided into 41 different fimbrial genotypes, and of these isolate, 285 strains (88.2%) had 12 to 14 fimbrial genes. Our findings indicated that the Salmonella isolates from different poultry species were phenotypically and genetically diverse and that some fimbrial genes are more frequently associated with serovars or serogroups.     

Register-based predictors of violations of animal welfare legislation in dairy herds

The assessment of animal welfare can include resource-based or animal-based measures. Official animal welfare inspections in Denmark primarily control compliance with animal welfare legislation based on resource measures (e.g. housing system) and usually do not regard animal response parameters (e.g. clinical and behavioural observations). Herds selected for welfare inspections are sampled by a risk-based strategy based on existing register data. The aim of the present study was to evaluate register data variables as predictors of dairy herds with violations of the animal welfare legislation (VoAWL) defined as occurrence of at least one of the two most frequently violated measures found at recent inspections in Denmark, namely (a) presence of injured animals not separated from the rest of the group and/or (b) animals in a condition warranting euthanasia still being present in the herd. A total of 25 variables were extracted from the Danish Cattle Database and assessed as predictors using a multivariable logistic analysis of a data set including 73 Danish dairy herds, which all had more than 100 cows and cubicle loose-housing systems. Univariable screening was used to identify variables associated with VoAWL at a P-value<0.2 for the inclusion in a multivariable logistic regression analysis. Backward selection procedures identified the following variables for the final model predictive of VoAWL: increasing standard deviation of milk yield for first lactation cows, high bulk tank somatic cell count (⩾250 000 cells/ml) and suspiciously low number of recorded veterinary treatments (⩽25 treatments/100 cow years). The identified predictors may be explained by underlying management factors leading to impaired animal welfare in the herd, such as poor hygiene, feeding and management of dry or calving cows and sick animals. However, further investigations are required for causal inferences to be established.     

Diagnostic Potential of Anti-rNcp-43 Polyclonal Antibodies for the Detection of Neospora caninum

Neosporosis is a disease caused by the apicomplexan parasite Neospora caninum, which is closely related to Toxoplasma gondii. N. caninum infection represents an important cause of reproductive failure in sheep, goats, horses, and cattle worldwide. The diagnosis of neosporosis is based on the detection of pathogen-specific antibodies in animal sera or the presence of tissue cysts. However, morphological similarities and serological cross-reactivity between N. caninum and T. gondii can result in the misdiagnosis. In this study, the N. caninum tachyzoite surface protein Ncp-43 was expressed in a recombinant form to elicit polyclonal antibodies (pAb) response. The pAb was purified and conjugated to horseradish peroxidase (HRP) or fluorescein isothiocyanate (FITC) to detect the recombinant and native Ncp-43 proteins, respectively. The pAb and pAb/HRP were able to recognize rNcp-43 by dot blot and ELISA, and pAb/FITC immunolabeled the apical complex of tachyzoites. A blocking enzyme-linked immunosorbent assay (b-ELISA) was performed to evaluate pAb/HRP as a diagnostic tool. The mean percent inhibition for the positive and negative serum samples from cattle with neosporosis was significantly different (P < 0.0001). These results suggest that the pAb may bind to the same epitopes of Ncp-43 as anti-N. caninum antibodies in the positive samples tested. The b-ELISA using the pAb/HRP can facilitate diagnostic testing for neosporosis, since fewer steps are involved, and cross-reactivity with secondary antibodies is avoided. In summary, this report describes the production of antibodies against N. caninum, and evaluates the potential of these tools for the development of new diagnostic tests for neosporosis.     

Fitness of Salmonella enterica serovar Thompson in the Cilantro Phyllosphere

The epiphytic fitness of Salmonella enterica was assessed on cilantro plants by using a strain of S. enterica serovar Thompson that was linked to an outbreak resulting from cilantro. Salmonella serovar Thompson had the ability to colonize the surface of cilantro leaves, where it was detected by confocal laser scanning microscopy (CLSM) at high densities on the veins and in natural lesions. The population sizes of two common colonizers of plant surfaces, Pantoea agglomerans and Pseudomonas chlororaphis, were 10-fold higher than that of the human pathogen on cilantro incubated at 22°C. However, Salmonella serovar Thompson achieved significantly higher population levels and accounted for a higher proportion of the total culturable bacterial flora on cilantro leaves when the plants were incubated at warm temperatures, such as 30°C, after inoculation, indicating that the higher growth rates exhibited by Salmonella serovar Thompson at warm temperatures may increase the competitiveness of this organism in the phyllosphere. The tolerance of Salmonella serovar Thompson to dry conditions on plants at 60% relative humidity was at least equal to that of P. agglomerans and P. chlororaphis. Moreover, after exposure to low humidity on cilantro, Salmonella serovar Thompson recovered under high humidity to achieve its maximum population size in the cilantro phyllosphere. Visualization by CLSM of green fluorescent protein-tagged Salmonella serovar Thompson and dsRed-tagged P. agglomerans inoculated onto cilantro revealed that the human pathogen and the bacterial epiphyte formed large heterogeneous aggregates on the leaf surface. Our studies support the hypothesis that preharvest contamination of crops by S. enterica plays a role in outbreaks linked to fresh fruits and vegetables.     

Identification of Novel Salmonella enterica Serovar Typhimurium DT104-Specific Prophage and Nonprophage Chromosomal Sequences among Serovar Typhimurium Isolates by Genomic Subtractive Hybridization

Genomic subtractive hybridization was performed between Salmonella enterica serovar Typhimurium LT2 and DT104 to search for novel Salmonella serovar Typhimurium DT104-specific sequences. The subtraction resulted mainly in the isolation of DNA fragments with sequence similarity to phages. Two fragments identified were associated with possible virulence factors. One fragment was identical to irsA of Salmonella serovar Typhimurium ATCC 14028, which is suggested to be involved in macrophage survival. The other fragment was homologous to HldD, an Escherichia coli O157:H7 lipopolysaccharide assembly-related protein. Five selected DNA fragments—irsA, the HldD homologue, and three fragments with sequence similarity to prophages—were tested for their presence in 17 Salmonella serovar Typhimurium DT104 isolates and 27 non-DT104 isolates by PCR. All five selected DNA fragments were Salmonella serovar Typhimurium DT104 specific among the serovar Typhimurium isolates tested. These DNA fragments can be useful for better detection and typing of Salmonella serovar Typhimurium DT104.     

Added dietary cobalt or vitamin B12, or injecting vitamin B12 does not improve performance or indicators of ketosis in pre- and post-partum Holstein-Friesian dairy cows

Vitamin B₁₂ is synthesised in the rumen from cobalt (Co) and has a major role in metabolism in the peri-paturient period, although few studies have evaluated the effect of the dietary inclusion of Co, vitamin B₁₂ or injecting vitamin B₁₂ on the metabolism, health and performance of high yielding dairy cows. A total of 56 Holstein-Friesian dairy cows received one of four treatments from 8 weeks before calving to 8 weeks post-calving: C, no added Co; DC, additional 0.2 mg Co/kg dry matter (DM); DB, additional 0.68 mg vitamin B₁₂/kg DM; IB, intra-muscular injection of vitamin B₁₂ to supply 0.71 mg/cow per day prepartum and 1.42 mg/cow per day post-partum. The basal and lactation rations both contained 0.21 mg Co/kg DM. Cows were weighed and condition scored at drying off, 4 weeks before calving, within 24 h of calving and at 2, 4 and 8 weeks post-calving, with blood samples collected at drying off, 2 weeks pre-calving, calving and 2, 4 and 8 weeks post-calving. Liver biopsy samples were collected from all animals at drying off and 4 weeks post-calving. Live weight changed with time, but there was no effect of treatment (P>0.05), whereas cows receiving IB had the lowest mean body condition score and DB the highest (P<0.05). There was no effect of treatment on post-partum DM intake, milk yield or milk fat concentration (P>0.05) with mean values of 21.6 kg/day, 39.6 kg/day and 40.4 g/kg, respectively. Cows receiving IB had a higher plasma vitamin B₁₂ concentration than those receiving any of the other treatments (P<0.001), but there was no effect (P>0.05) of treatment on homocysteine or succinate concentrations, although mean plasma methylmalonic acid concentrations were lower (P=0.019) for cows receiving IB than for Control cows. Plasma β-hydroxybutyrate concentrations increased sharply at calving followed by a decline, but there was no effect of treatment. Similarly, there was no effect (P>0.05) of treatment on plasma non-esterified fatty acids or glucose. Whole tract digestibility of DM and fibre measured at week 7 of lactation were similar between treatments, and there was little effect of treatment on the milk fatty acid profile except for C15:0, which was lower in cows receiving DC than IB (P<0.05). It is concluded that a basal dietary concentration of 0.21 mg Co/kg DM is sufficient to meet the requirements of high yielding dairy cows during the transition period, and there is little benefit from additional Co or vitamin B₁₂.     

Isolation of Salmonella enterica Serovar Enteritidis from Houseflies (Musca domestica) Found in Rooms Containing Salmonella Serovar Enteritidis-Challenged Hens

Houseflies (Musca domestica) released into rooms containing hens challenged with Salmonella enterica serovar Enteritidis (Salmonella serovar Enteritidis) rapidly became contaminated with Salmonella serovar Enteritidis. Forty to 50% of the flies were contaminated at 48 h, and the percentage increased to 50 to 70% at 4 and 7 days postexposure and then decreased to 30% at day 15. Initial attempts at recovering surface organisms for culture using an aqueous rinse were largely unsuccessful, while cultures of internal contents readily recovered Salmonella serovar Enteritidis. However, when 0.5% detergent was incorporated into the rinse, high recovery levels of bacteria were observed from both external and internal culture regimens, indicating equal distribution of the organism on and in the fly and a tighter interaction of the organism with the host than previously thought. Salmonella serovar Enteritidis was isolated routinely from the fly gut, on rare occasions from the crop, and never from the salivary gland. Feeding contaminated flies to hens resulted in gut colonization of a third of the birds, but release of contaminated flies in a room containing previously unchallenged hens failed to result in colonization of any of the subject birds. These results indicate that flies exposed to an environment containing Salmonella serovar Enteritidis can become colonized with the organism and might serve as a source for transmission of Salmonella serovar Enteritidis within a flock situation.