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Short Communication

Short Communication Detection of Chlamydia psittaci in Belgian cattle with signs of respiratory disease and milk drop syndrome H. Van Loo, B. Pardon, P. De Schutter, K. De Bleecker, D. Vanrompay, P. Deprez, J. Maris THE term ‘milk drop syndrome’ is commonly used to refer to a sudden drop in milk yield in dairy cattle that may or may not be showing signs of other disease (Gunning and others 1999). According to Radostits and others (2007), milk drop syndrome is a herd syndrome in which the milk yield falls precipitately without any clear clinical evidence of disease or deprivation of food or water. Leptospirosis (caused by Leptospira Hardjo), heat stress and summer fescue toxicosis are among the more common causes (Radostits and others 2007). In recent years, multiple outbreaks of milk drop syndrome with fever have been reported in dairy cattle in several European countries (Crawshaw and others 2008, Guyot and others 2011). Bovine viral diarrhoea virus (BVDV) (Moerman and others 1994), Leptospira Hardjo (Pearson and others 1980), Anaplasma phagocytophilum (De Schutter and others 2011, Guyot and others 2011) and Schmallenberg virus (Veldhuis and others 2013) have been identified in several of these case herds. A potential role of influenza A virus has also been suggested (Brown and others 1998, Gunning and others 1999, Crawshaw and others 2008). Within the context of the Flemish cattle health monitoring programme (Veepeiler Rund, Belgium), a substantial number of herds recently affected with milk drop syndrome remained undiagnosed after being tested for all of the aforementioned pathogens. Additionally, respiratory symptoms were frequently mentioned as clinical signs on these farms. Since Chlamydia psittaci has recently been identified as being involved in reproductive disorders and airway inflammation in calves (Reinhold and others 2011, Ostermann and others 2013), this short communication aims to report on clinical and environmental findings in seven dairy herds with milk drop Veterinary Record (2014) H. Van Loo, DVM, K. De Bleecker, DVM, J. Maris, DVM, Animal Health Center Flanders (DGZ Vlaanderen), Industrielaan 29, Torhout 8820, Belgium B. Pardon, DVM, Ph.D, P. De Schutter, DVM, P. Deprez, DVM, Ph.D, Dipl. ECBHM, Department of Large Animal Internal Medicine, Faculty of Veterinary Medicine, Ghent University,

doi: 10.1136/vr.102527 Salisburylaan 133, Merelbeke 9820, Belgium D. Vanrompay, DVM, Ph.D, Department of Molecular Biotechnology, Ghent University, Coupure links 653, Ghent 9000, Belgium E-mail for correspondence: [email protected] Provenance: not commissioned; externally peer reviewed Accepted October 9, 2014

syndrome with respiratory symptoms where C. psittaci was detected in bronchoalveolar lavage (BAL) samples from diseased cows. Between June 2011 and February 2014, 21 dairy herds with respiratory symptoms and milk drop were visited for an extensive diagnostic assessment. Initially (between June 2011 and September 2011), C. psittaci was not included in the analysis protocol (since the differential list was already rather extensive and a causative role for C. psittaci in dairy cattle with respiratory distress was not as well documented at the time). As well as consistent reports of nasal discharge, fever, tachypnoea and cough in the first 11 herds visited, the animals showed one or more of the following signs, with large variation between herds: milk drop (0–30 per cent loss of milk), reddening of the nose and teats, diarrhoea, abortion, anorexia, mastitis and limb oedema. In two of the 11 herds visited, A. phagocytophilum was found by means of seroconversion (A. phagocytophilum Ac. in-house indirect immunofluorescence test, Laboratoire de developpement et d’analyses zoopole Plouffragan, France) and/or PCR (Kit TaqVet, Laboratoire Service International, France). In two of the other herds, recent contact with BVDV was demonstrated by seroconversion (SERELISA BVD p80 Ab Mono Blocking, Synbiotics, France) and/or by BVDV antigen detection (Bovine Viral Diarrhoea Virus Antigen Test Kit/Serum Plus, Idexx, Switzerland). Convalescent blood samples of affected animals were also analysed for bovine adenovirus 3 (ELISA kit for Bovine Adenovirus 3, Bio-X Diagnostics, Belgium), bovine respiratory syncytial virus (ELISA kit for BRSV, Bio-X Diagnostics), bovine herpes virus 1 (ELISA kit for IBR, Bio-X Diagnostics), Mycoplasma bovis (ELISA kit for M. bovis, Bio-X Diagnostics) and para-influenza virus 3 (ELISA kit for Parainfluenza 3, Bio-X Diagnostics). However, seroconversion rates were too low to conclude that there was any biological causation (data not shown). Cell cultures (of Madin-Darby bovine kidney cell lines) were incubated with BAL samples from three diseased animals per herd. No viral replication or cytopathogenic effects of influenza A virus were detected. Furthermore, after the Schmallenberg virus outbreak in late summer 2011, the stored sera from the 11 affected herds were retrospectively investigated by real time RT-PCR (Veterinary and Agrochemical Research Centre, Belgium), but none were positive. Considering the absence of typical symptoms of leptospirosis and the finding that none of the dams, which occasionally aborted in 18 of the 21 included herds, tested seropositive (within the context of the official obligatory Belgian abortion surveillance programme by PrioCHECK L. hardjo Ab, Prionics, Switzerland) in the same time frame, testing for Leptospira Hardjo was not systematically performed after visiting the affected herds. With this diagnostic approach, more than 60 per cent of these problem herds remained undiagnosed. Therefore, from December 2012 onwards, a nested PCR, specific for C. psittaci (Van Loock and others 2005), was performed on BAL samples from 10 newly affected herds. Clinical findings and general herd information on these 10 herds are provided in Table 1. All but one herd was located in the north-eastern part of Belgium (Antwerp and Limburg). The most prominent clinical findings in these herds were respiratory signs (serous nasal discharge, cough and tachypnoea), fever (39.2–41.0°C) and milk drop syndrome affecting 7–100 per cent of the lactating animals within a period of one to two weeks. BAL samples were taken from two to five diseased dairy cows per herd as described previously (Catry and others 2008). BAL samples were transported in DNA/RNA stabilisation buffer (Roche) at 4°C for analysis the next day. Samples from Herd 2 were additionally transported in Chlamydia transport medium and stored at −80°C for subsequent culture in Buffalo-Green-Monkey (BGM) cells. In seven of these 10 (70 per cent) herds, positive PCR results on BAL December 6, 2014 | Veterinary Record

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Short Communication TABLE 1: Laboratory diagnostic information and environmental factors of 10 dairy herds with respiratory disease and milk drop in adult cattle, reported in 2013 Herd

Period of clinical signs

1 2 3 4 5 6 7 8 9 10

August 2012–October 2013 Sept 2012–August 2013 March–April 2013 March–April 2013 July–August 2013 July–October 2013 September–November 2013 September–November 2013 October 2013 October 2013–February 2014

% Affected animals

Estimated % milk loss

50 60–70 ? 85 7 100 90–100 80–90 30–40 50

? 10–15 10–20 10 10–15 0 10 20–30 10–15 20

IBR status Certified free Vaccination 2×/year Vaccination 2×/year Vaccination 2×/year Vaccination 2×/year Vaccination 2×/year Certified free Certified free Vaccination 2×/year Vaccination 2×/year

BVDV status (Ag detection)

Chlamydia psittaci PCR % positives (number)*

Zero grazing Y/N

Negative Infected Infected Negative Negative Negative Negative Negative Infected Negative

0 (0/4) 50 (2/4) 66 (2/3) 50 (1/2) 33 (1/3) 25 (1/4) 25 (1/4) 0 (0/5) 0 (0/2) 25 (1/4)


*All sampled animals showed clinical symptoms BVDV, bovine viral diarrhoea virus; IBR, infectious bovine rhinotracheitis.

samples were obtained in 25–66 per cent of the sampled animals (Table 1). Furthermore, in Herd 2, the presence of C. psittaci was also confirmed by isolation in BGM cells, resulting in two (out of three) samples testing positive. Afterwards, in this herd, two environmental samples were taken, and both (air and water) samples were PCR positive. Of all other pathogens tested, only BVDV circulation was detected in three of 10 (30 per cent) herds. Remarkably, six of seven C. psittaci-infected herds were not grazed (housed all year round) (Table 1). As in other countries, C. psittaci is highly prevalent in domesticated and wild birds in Belgium (Vanrompay and others 1997, Dickx and others 2010, 2013). Despite its ubiquitous presence in cattle, as is the case in several European countries (Pospischil and others 2002, Borel and others 2006, Kauffold and others 2007, Teankum and others 2007, Kemmerling and others 2009), C. psittaci has not been detected nor looked for previously in Belgian cattle. Bovine chlamydial infections have been associated with a wide range of (sub)clinical presentations, of which reduced milk production, mastitis, fertility disorders, abortion and premature calving are most frequently reported in adult cattle, whereas perinatal death and respiratory disease are reported in calves (Wehrend and others 2005, Kemmerling and others 2009, Reinhold and others 2011). C. psittaci has been linked to respiratory disease in calves, both after natural and experimental infection (Ostermann and others 2013). To the authors’ knowledge, there is only one report that suggests a possible role for C. psittaci in respiratory disease in adult dairy cattle (Dannatt and others 1998). As well as respiratory disease and milk drop syndrome, the previous report mentioned a substantial increase in abortion rates, whereas this was not seen in the present case series. A possible explanation might be the involvement of Chlamydophila abortus in the outbreaks described by Dannatt and others (1998), since the PCR test (used in those cases) was originally designed to identify ruminant abortion and psittacine strains of C. psittaci (Hewinson and others 1997). After reclassification of the Chlamydiales, it was claimed that the PCR test used in that study did not differentiate between C. psittaci and C. abortus (Everett and others 1999). Another difference between the present report and the one of Dannatt and others (1998) is the detection of C. psittaci in BAL samples of diseased dairy cattle. Dannatt and others (1998) were not able to demonstrate the pathogen in samples of the upper respiratory tract (only in blood and fetal tissue). A potential role for Chlamydiae in multifactorial diseases in dairy cattle and calves has previously been suggested (Reinhold and others 2011). However, the possible interactions with other pathogens or environmental factors and the exact contribution of Chlamydiae in the pathogenesis of respiratory disease are currently insufficiently clarified. The detection of C. psittaci DNA in BAL samples of dairy cattle with respiratory disease is, on its own, insufficient to conclude that this pathogen is a causative agent of respiratory Veterinary Record | December 6, 2014

disease and milk drop syndrome. Therefore, case-control studies are necessary to clarify the pathogenesis of clinical C. psittaci infection, next to observational studies identifying risk factors for C. psittaci infection in the herd. It was remarkable that most of the C. psittaci-positive herds (six of seven) had no grazing management system in place, in contrast to the majority of cattle farms in Belgium. The demonstrated presence of C. psittaci in environmental samples of dairy herds might lead—in zero grazing conditions—to increased infection pressure due to close contact between the animals and year-round occupation of the stables. A thorough risk factor analysis should be performed, comparing different management systems to elucidate the potential increased risk for clinical C. psittaci infection in cattle that are not grazed, as well as the potential zoonotic risk. Since the presence of wild or domesticated birds was reported on several of the affected farms, it could be worthwhile to include this parameter in future studies as well. Geographical effects should also be taken into account, as most of the C psittacipositive herds in this study were located in the provinces of Antwerp and Limburg, which is a prominent dairy region in Belgium. Known risk factors for infection with Chlamydia species in cattle are the use of replacement animals from external sources, use of breeding bulls, absence of individual calving pens and poor hygiene in the cow environment (Kemmerling and others 2009). In conclusion, after three years of extensive laboratory investigations into field outbreaks of respiratory diseases in adult dairy cattle, a large proportion remained undiagnosed after the exclusion of known respiratory pathogens commonly encountered in Europe. In contrast to this, the detection of C. psittaci in predominantly zero grazing dairy herds displaying respiratory disease and milk drop syndrome might be a valuable contribution to ongoing research into the potential multifactorial pathogenesis of C. psittaci infections in dairy cattle, which merits further attention.

Acknowledgements The authors acknowledge both farmers and local practitioners for their kind cooperation. Funding This work was financially supported by the Flemish Cattle Health Monitoring Programme (Veepeiler Rund).

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Detection of Chlamydia psittaci in Belgian cattle with signs of respiratory disease and milk drop syndrome H. Van Loo, B. Pardon, P. De Schutter, K. De Bleecker, D. Vanrompay, P. Deprez and J. Maris Veterinary Record 2014 175: 562 originally published online October 28, 2014

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Detection of Chlamydia psittaci in Belgian cattle with signs of respiratory disease and milk drop syndrome.

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