Detection of Babesia divergens in southern Norway by using an immunofluorescence antibody test in cow sera

Background

The incidence of bovine babesiosis, caused by Babesia divergens (Apicomplexa: Piroplasmida) has decreased markedly since the 1930 s, but may re-emerge as a consequence of climate change and changes in legislation and pasturing practices. This is a potentially serious disease, with both economical and animal welfare consequences. Therefore, there is a need to survey the distribution of B. divergens.

Methods

We tested sera from 306 healthy pastured cows from 24 farms along the southern Norwegian coast by using an indirect immunofluorescence IgG antibody test (IFAT). Fractions of seropositive cows were compared by calculating 95% CI.

Results

The results of this test showed that 27% of the sera were positive for B. divergens antibodies. The fraction of antibody-positive sera that we detected showed a two-humped distribution, with a high fraction of positives being found in municipalities in the western and eastern parts of the study area, while the municipalities between these areas had few or no positive serum samples.

Conclusions

Neither the farmers'' observations nor the Norwegian Dairy Herd Recording System give an adequate picture of the distribution of bovine babesiosis. Serological testing of cows by using IFAT is a convenient way of screening for the presence of B. divergens in an area.     

A comparative study of clinical manifestations,haematological and serological responses after experimental infection with Anaplasma phagocytophilum in two Norwegian sheep breeds

Background

It has been questioned if the old native Norwegian sheep breed, Old Norse Sheep (also called Norwegian Feral Sheep), normally distributed on coastal areas where ticks are abundant, is more protected against tick-borne infections than other Norwegian breeds due to a continuously high selection pressure on pasture. The aim of the present study was to test this hypothesis in an experimental infection study.

Methods

Five-months-old lambs of two Norwegian sheep breeds, Norwegian White (NW) sheep and Old Norse (ON) sheep, were experimentally infected with a 16S rRNA genetic variant of Anaplasma phagocytophilum (similar to GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"M73220","term_id":"148293"}}M73220). The experiment was repeated for two subsequent years, 2008 and 2009, with the use of 16 lambs of each breed annually. Ten lambs of each breed were inoculated intravenously each year with 0.4 ml A. phagocytophilum-infected blood containing approximately 0.5 × 10⁶ infected neutrophils/ml. Six lambs of each breed were used as uninfected controls. Half of the primary inoculated lambs in each breed were re-challenged with the same infectious dose at nine (2008) and twelve (2009) weeks after the first challenge. The clinical, haematological and serological responses to A. phagocytophilum infection were compared in the two sheep breeds.

Results

The present study indicates a difference in fever response and infection rate between breeds of Norwegian sheep after experimental infection with A. phagocytophilum.

Conclusion

Although clinical response seems to be less in ON-lambs compared to NW-lambs, further studies including more animals are needed to evaluate if the ON-breed is more protected against tick-borne infections than other Norwegian breeds.     

Anaplasma phagocytophilum in Danish sheep: confirmation by DNA sequencing

Background

The presence of Anaplasma phagocytophilum, an Ixodes ricinus transmitted bacterium, was investigated in two flocks of Danish grazing lambs. Direct PCR detection was performed on DNA extracted from blood and serum with subsequent confirmation by DNA sequencing.

Methods

31 samples obtained from clinically normal lambs in 2000 from Fussingø, Jutland and 12 samples from ten lambs and two ewes from a clinical outbreak at Feddet, Zealand in 2006 were included in the study. Some of the animals from Feddet had shown clinical signs of polyarthritis and general unthriftiness prior to sampling. DNA extraction was optimized from blood and serum and detection achieved by a 16S rRNA targeted PCR with verification of the product by DNA sequencing.

Results

Five DNA extracts were found positive by PCR, including two samples from 2000 and three from 2006. For both series of samples the product was verified as A. phagocytophilum by DNA sequencing.

Conclusions

A. phagocytophilum was detected by molecular methods for the first time in Danish grazing lambs during the two seasons investigated (2000 and 2006).     

Changing incidence of bovine babesiosis in Ireland

Background

In Ireland bovine babesiosis is caused by the tick-borne blood parasite, Babesia divergens. A survey of veterinary practitioners and farmers in the 1980’s revealed an annual incidence of 1.7% associated with considerable economic losses. However, two subsequent surveys in the 1990’s indicated a decline in clinical babesiosis. Recent evidence from continental Europe suggests that, probably due to climate change, the distribution of the tick vector of B. divergens, Ixodes ricinus is extending to more northerly regions and higher altitudes. In addition, milder winters are thought to widen the window of tick activity.In order to determine whether any such changes have affected the incidence of bovine babesiosis in Ireland, a questionnaire survey of farmers and veterinarians was carried out and compared with data from previous surveys.

Results

Our survey indicates that while the incidence of clinical disease has continued to decline, cases can occur at any time of year. In contrast to previous surveys, affected farms were the same size as unaffected ones. There was no correlation between disease risk and the presence of deer on the land. Disease severity and mortality rates were increased because many infections were advanced by the time they were detected and treated.

Conclusion

While the precise reasons for the decline in the incidence of redwater are unknown, changes in agricultural practice are likely to be of importance. A reversal of the trend could be devastating, as vigilance among farmers and veterinarians is flagging and the national herd is losing its protective immunity to disease.     

A morphological and molecular study of Anaplasma phagocytophilum transmission events at the time of Ixodes ricinus tick bite

Background

Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA) in humans and tick-borne fever (TBF) in ruminants. The bacterium invades and replicates in phagocytes, especially in polymorphonuclear granulocytes.

Methods

In the present study, skin biopsies and ticks (Ixodes ricinus) were collected from tick feeding lesions on 38 grazing lambs between two and three weeks after access to pastures. The histopathological changes associated with tick bites and A. phagocytophilum infection, were described. In addition the skin biopsies were examined by immunohistochemistry. Furthermore, samples from blood, skin biopsies and ticks were examined by serology, PCR amplification of msp2 (p44), genotyping of rrs (16S rRNA) variants, and compared with the results obtained from histological and immunohistochemical investigations.

Results

Tick bites were associated with chronic and hyperplastic inflammatory skin lesions in this study. A. phagocytophilum present in skin lesions were mainly associated with neutrophils and macrophages. Bacteria were occasionally observed in the Tunica media and Tunica adventitia of small vessels, but were rarely found in association with endothelial cells. PCR and genotyping of organisms present in blood, ticks and skin biopsies suggested a haematogenous and a local spread of organisms at the tick attachment sites.

Conclusions

The present study describes different aspects of A. phagocytophilum infection at the site of tick bite, and indicates that A. phagocytophilum rarely associates with endothelium during the early pathogenesis of infection.     

Screening for several potential pathogens in feral pigeons (Columba livia) in Madrid

Background

Pathogens with the zoonotic potential to infect humans, such as Campylobacter jejuni, Campylobacter coli and Chlamydophila psittaci, can be found in feral pigeons (Columba livia). Given the high density of these birds in the public parks and gardens of most cities, they may pose a direct threat to public health.

Methods

A total of 118 pigeons were captured in three samplings carried out in 2006-2007 in public parks and gardens in Madrid, Spain. Standard haematological and morphological analyses were carried out on the pigeons. PCR was used to screen for the presence of Campylobacter jejuni, C. coli and Chlamydophila psittaci. Positive samples were confirmed by DNA sequencing.

Results

The analyses demonstrated a high prevalence of Chlamydophila psittaci (52.6%) and Campylobacter jejuni (69.1%) among the birds captured. In contrast, Campylobacter coli was rarely detected (1.1%).

Conclusions

Pigeons in Madrid can carry Chlamydophila psittaci and Campylobacter jejuni. They may be asymptomatic or subclinical carriers of both pathogens.     

Health status of seabirds and coastal birds found at the German North Sea coast

Background

Systematic pathological investigations to assess the health status of seabirds and coastal birds in Germany were performed. The investigation was conducted to obtain data on possible causes of decline in seabird and coastal bird populations.

Methods

48 individuals of 11 different species of seabirds and coastal birds were collected by the stranding network along the entire German North Sea coast from 1997 to 2008, including mainly waders such as Eurasian oystercatchers (Haematopus ostralegus) and red knots (Calidris canutus) as well as seabirds such as northern fulmars (Fulmaris glacialis) and common scoters (Melanitta nigra).For most birds (n = 31) found dead along the shore no obvious cause of death was evident, while 17 individuals were killed by collisions with lighthouses.

Results

Overall, the nutritional status of the investigated birds was very poor, and the body mass in most cases was significantly lower compared to masses of living birds caught during the same periods of the year. This is partly linked to chronic parasitic or bacterial infections in different organs or to septicaemia. In some cases infections with zoonotic tuberculosis caused by Mycobacterium spp. were found. Avian influenza was not found in any of the collected birds.

Conclusion

The presented data contribute to the evaluation of the health status of birds in the German North Sea. Moreover, they present an important tool for the assessment of potential pathogens with an impact on the health status of seabirds and coastal birds.     

Seroprevalence of Toxoplasma gondii infection in Norwegian dairy goats

Background

Toxoplasma gondii is a major problem for the sheep industry as it may cause reproduction problems. The importance of T. gondii in Norwegian goat herds is uncertain, but outbreaks of toxoplasmosis in dairy goat farms have been recorded. The aim of this study was to describe the prevalence of T. gondii infection in Norwegian dairy goats by using serology.

Findings

Goat serum originally collected as part of two nationwide surveillance and control programmes between 2002 and 2008 were examined for T. gondii antibodies by using direct agglutination test. In total, 55 of 73 herds (75%) had one or more serologically positive animals, while 377 of 2188 (17%) of the individual samples tested positive for T. gondii antibodies.

Conclusions

This is the first prevalence study of T. gondii infection in Norwegian goats. The results show that Norwegian goat herds are commonly exposed to T. gondii. Nevertheless, the majority of goat herds have a low prevalence of antibody positive animals, which make them vulnerable to infections with T. gondii during the gestation period.     

Investigation of Chlamydophila spp. in dairy cows with reproductive disorders

Background

Reports worldwide indicate high prevalence of Chlamydophila spp. infection in cattle. To assess the prevalence in Sweden, 525 cows in 70 dairy herds with reproductive disorders was investigated.

Methods

To detect antibodies two commercially available kits were used. Moreover, 107 specimens, including vaginal swabs, organ tissues and milk were analysed by Polymerase Chain Reaction (PCR).

Results

Two (0.4%) cows were seropositive in the Pourquier Cp. abortus ELISA. The seroprevalence with the Chekit ELISA was 28% with no difference between cases and controls. Five specimens were positive in real-time PCR and further analysed by nested PCR. Cp. pecorum was confirmed by partial omp1 DNA sequencing of the nested PCR product of vaginal swabs from control cows.

Conclusion

The results suggest that Cp. abortus infection is absent or rare in Swedish cows whereas Cp. pecorum is probably more spread. They also suggest that Chlamydophila spp. are not related to reproduction disorders in Swedish cattle.     

Prevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in questing Ixodes ricinus ticks in relation to the density of wild cervids

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum have been considered as pathogens in animals and humans. The role of wild cervids in the epidemiology is not clear. We analyzed questing Ixodes ricinus ticks collected in spring for these pathogens from sites with high (Fjelløyvær and Strøm) and low density (Tjore, Hinnebu and Jomfruland) of wild cervids to study the spread of the pathogens in questing ticks.

Methods

For detection of Anaplasma phagocytophilum a 77-bp fragment in the msp2 gene was used. Detection of Borrelia burgdorferi sensu lato was performed using the FL6 and FL7 primers according to sequences of conserved regions of the fla gene. The OspA gene located on the linear 49-kb plasmid was used as target in multiplex PCR for genotyping. Genospecies-specific primers were used in the PCR for Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii.

Results

Infection rates with Borrelia spp. were significantly lower at Fjelløyvær and Strøm compared to Tjore and Hinnebu; Fjelløyvær vs. Tjore (χ² = 20.27, p < 0.0001); Fjelløyvær vs. Hinnebu (χ² = 24.04, p < 0.0001); Strøm vs. Tjore (χ² = 11.47, p = 0.0007) and Strøm vs. Hinnebu (χ² = 16.63, p < 0.0001). The Borrelia genospecies were dominated by. B. afzelii (82%) followed by B. garinii (9.7%) and B. burgdorferi sensu stricto (6.9%). B. burgdorferi s.s. was only found on the island of Jomfruland. The infection rate of Anaplasma phagocytophilum showed the following figures; Fjelløyvær vs Hinnebu (χ² = 16.27, p = 0.0001); Strøm vs. Tjore (χ² = 13.16, p = 0.0003); Strøm vs. Hinnebu (χ² = 34.71, p < 0.0001); Fjelløyvær vs. Tjore (χ² = 3.19, p = 0.0742) and Fjelløyvær vs. Støm (χ² = 5.06, p = 0.0245). Wild cervids may serve as a reservoir for A. phagocytophilum. Jomfruland, with no wild cervids but high levels of migrating birds and rodents, harboured both B. burgdorferi s.l. and A. phagocytophilum in questing I. ricinus ticks. Birds and rodents may play an important role in maintaining the pathogens on Jomfruland.

Conclusion

The high abundance of roe deer and red deer on the Norwegian islands of Fjelløyvær and Strøm may reduce the infection rate of Borrelia burgdorferi sensu lato in host seeking Ixodes ricinus, in contrast to mainland sites at Hinnebu and Tjore with moderate abundance of wild cervids. The infection rate of Anaplasma phagocytophilum showed the opposite result with a high prevalence in questing ticks in localities with a high density of wild cervids compared to localities with lower density.     

Prevalence of Anaplasma phagocytophilum infection and effect on lamb growth

Background

A major challenge in sheep farming during the grazing season along the coast of south-western Norway is tick-borne fever (TBF) caused by the bacteria Anaplasma phagocytophilum that is transmitted by the tick Ixodes ricinus.

Methods

A study was carried out in 2007 and 2008 to examine the prevalence of A. phagocytophilum infection and effect on weaning weight in lambs. The study included 1208 lambs from farms in Sunndal Ram Circle in Møre and Romsdal County in Mid-Norway, where ticks are frequently observed. All lambs were blood sampled and serum was analyzed by an indirect fluorescent antibody assay (IFA) to determine an antibody status (positive or negative) to A. phagocytophilum infection. Weight and weight gain and possible effect of infection were analyzed using ANOVA and the MIXED procedure in SAS.

Results

The overall prevalence of infection with A. phagocytophilum was 55%. A lower weaning weight of 3% (1.34 kg, p < 0.01) was estimated in lambs seropositive to an A. phagocytophilum infection compared to seronegative lambs at an average age of 137 days.

Conclusions

The results show that A. phagocytophilum infection has an effect on lamb weight gain. The study also support previous findings that A. phagocytophilum infection is widespread in areas where ticks are prevalent, even in flocks treated prophylactic with acaricides.     

Prophylactic treatment with flumethrin,a pyrethroid (Bayticol?, Bayer), against Anaplasma phagocytophilum infection in lambs

Backgroud

Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) causes the disease tick-borne fever (TBF) in domestic ruminants and has for decades been one of the main scourges for the sheep industry in the coastal areas of Norway. Current control strategies are based on reduction of tick infestation by chemical acaricides.

Methods

In the present study, we investigated if frequent pour-on applications of pyrethroids would reduce tick infestion rate and seroprevalence of A. phagocytophilum infection in sheep. Forty lambs, one month old, of the Norwegian White Sheep breed were used. The lambs belonged to the experimental sheep flock at the Department of Production Animal Clinical Sciences. None of the lambs had been on I. ricinus infested pasture before turnout (day 0). All lambs were twins and twenty lambs were treated with a pour-on pyrethroid (Bayticol®, Bayer A/S, DK-2300) with a dose of 5 ml on days 0, 14, 28, 42, 56, 70, 84, 98, 112 and 128. Twenty lambs were untreated controls. The lambs were collected every fourteen days on pasture for treatment. In addition, the lambs were examined for ticks, blood sampled, weighed, and rectal temperature was recorded.

Results and conclusion

A significant reduction in tick infestion rate was detected on treated lambs. However, the present results indicate that frequent acaricide treatment does not reduce the seroprevalence to A. phagocytophilum on tick-infested pasture.     

Investigation of Chlamydiaceae in semen and cauda epididymidis and seroprevalence of Chlamydophila abortus in breeding bulls

Background

Reproductive disorders associated with chlamydial infection have been reported worldwide in cattle and there are indications of potential venereal transmission.

Methods

Semen samples from 21 dairy bulls and cauda epididymidis tissue samples from 43 beef bulls were analysed for chlamydial agent by real-time polymerase chain reaction (PCR) including an internal amplification control (mimic). Additionally, presence of antibodies against Chlamydophila (Cp.) abortus among the bulls was investigated with the commercial Pourquier^® ELISA Cp. abortus serum verification kit.

Results

No chlamydial agent was detected by PCR in either the semen samples or in the tissue samples. Additionally, no antibodies against Cp. abortus were detected.

Conclusions

The results suggest that Cp. abortus is very rare, or absent in Swedish bulls and thus the risk for venereal transmission of chlamydial infection through their semen is low. However, because Chlamydophila spp. infection rates seem to differ throughout the world, it is essential to clarify the relative importance of transmission of the infection through semen on cattle fertility.     

An investigation on the presence of Chlamydiaceae in Swedish dogs

Background

Bacteria belonging to the family Chlamydiaceae cause a broad spectrum of diseases in a wide range of hosts, including man, other mammals, and birds. Upper respiratory and genital diseases are common clinical problems caused by Chlamydiaceae. Very little is known about chlamydial infections in dogs. Few clinical reports on natural disease in dogs describe mainly conjunctival and upper respiratory signs, and the role of Chlamydiaceae in genital disease is unclear. The present study aimed at studying the prevalence of Chlamydiaceae in healthy dogs and in dogs with genital or upper respiratory disease, including conjunctivitis.

Methods

A real-time polymerase chain reaction (PCR) for Chlamydiaceae was used to detect any chlamydial species within this family. Swab samples from the conjunctiva and the mucosal membranes of the oropharynx, rectum and genital tract were taken from 79 dogs: 27 clinically healthy dogs, 25 dogs with clinical signs from the genital tract and 28 dogs with conjunctivitis. There were 52 female and 27 male dogs. From 7 of the male dogs, additional semen samples were analysed.

Results

No Chlamydiaceae were detected from any dog.

Conclusions

Although the number of dogs that was included is limited, the results suggest that cases of Chlamydiaceae in dogs probably are related to infection from other species, and that dogs in general do not harbour Chlamydiaceae. Bacteria belonging to the family Chlamydiaceae do not seem to be of major importance for genital or ocular disease in Swedish dogs.     

Superinfection occurs in Anaplasma phagocytophilum infected sheep irrespective of infection phase and protection status

Background

Anaplasma phagocytophilum infection in domestic ruminants is widespread in the coastal areas of southern Norway. The bacteria may persist in mammalian hosts. Several genetic variants of A. phagocytophilum exist. In the present study, we investigate whether superinfection occurs in the acute and persistent phase of the infection.

Methods

Five-month-old lambs of the Norwegian Dala breed were experimentally infected with two 16S rRNA gene variants of A. phagocytophilum, i.e. A. phagocytophilum variant 1 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"M73220","term_id":"148293","term_text":"M73220"}}M73220) and variant 2 (GenBank acc. no. {"type":"entrez-nucleotide","attrs":{"text":"AF336220","term_id":"13325085","term_text":"AF336220"}}AF336220). Eighteen lambs were used, two lambs in each group. Eight groups were experimentally inoculated with either variant 1 or 2 on day 0. Six of these groups were then challenged with the other variant on either days 7, 42 or 84, respectively. One group was left uninfected. The occurrence of A. phagocytophilum in blood samples was determined using semi-nested PCR analysis and gene sequencing. Specific antibodies were measured by an indirect immunofluorescence antibody assay (IFA).

Results

A. phagocytophilum variant 1 and 2 differed significantly with regards to clinical reaction and cross-immunity in infected lambs. Both variants were found in the blood after challenge. However, variant 1 was detected most frequently.

Conclusion

The present experiment indicates that superinfection of different genotypes occurs during the acute as well as the persistent phase of an A. phagocytophilum infection, even in lambs protected against the challenged infection.     

Benzimidazole resistance of sheep nematodes in Norway confirmed through controlled efficacy test

Background

Resistance against benzimidazoles (BZ) has recently been detected in Norwegian sheep flocks through a large scale prevalence survey based on the faecal egg count reduction test (FECRT). The use of this test in combination with bulk larval culture only gives an indication of which gastrointestinal nematodes genera that are involved and these results have to be confirmed by a controlled efficacy test (CET) to get accurate information about resistant nematodes populations at species level. A CET was therefore performed with larvae from two flocks where BZ resistance was previously detected through FECRT.

Results

The latter test confirmed the previous results in both flocks. In flock A, the BZ resistant nematode population consisted solely of Haemonchus contortus, whereas H. contortus and Teladorsagia circumcincta comprised the resistant worm population in flock B.

Conclusions

Some discrepancies that have been recorded between FECRT and CET results regarding time for post-treatment coproscopical examination and a temporary suppression of faecal egg excretion are discussed.     

A longitudinal study of Campylobacter distribution in a turkey production chain

Background

Campylobacter is the most common cause of bacterial enteritis worldwide. Handling and eating of contaminated poultry meat has considered as one of the risk factors for human campylobacteriosis.Campylobacter contamination can occur at all stages of a poultry production cycle. The objective of this study was to determine the occurrence of Campylobacter during a complete turkey production cycle which lasts for 1,5 years of time. For detection of Campylobacter, a conventional culture method was compared with a PCR method. Campylobacter isolates from different types of samples have been identified to the species level by a multiplex PCR assay.

Methods

Samples (N = 456) were regularly collected from one turkey parent flock, the hatchery, six different commercial turkey farms and from 11 different stages at the slaughterhouse. For the detection of Campylobacter, a conventional culture and a PCR method were used. Campylobacter isolates (n = 143) were identified to species level by a multiplex PCR assay.

Results

No Campylobacter were detected in either the samples from the turkey parent flock or from hatchery samples using the culture method. PCR detected Campylobacter DNA in five faecal samples and one fluff and eggshell sample. Six flocks out of 12 commercial turkey flocks where found negative at the farm level but only two were negative at the slaughterhouse.

Conclusion

During the brooding period Campylobacter might have contact with the birds without spreading of the contamination within the flock. Contamination of working surfaces and equipment during slaughter of a Campylobacter positive turkey flock can persist and lead to possible contamination of negative flocks even after the end of the day''s cleaning and desinfection. Reduction of contamination at farm by a high level of biosecurity control and hygiene may be one of the most efficient ways to reduce the amount of contaminated poultry meat in Finland. Due to the low numbers of Campylobacter in the Finnish turkey production chain, enrichment PCR seems to be the optimal detection method here.