In 1967, Rofe described her findings from an investigation of an August 1963 outbreak of perinatal mortality in maiden ewes near Goulburn. On finding pathology changes consistent with 'enzootic abortion' she remarked:
'The occurrence of enzootic abortion of ewes (EAE) in Australia has been suspected for some years, and serological evidence of the presence of the psittacosis lymphogranuloma venereum group was detected in a high proportion of 430 sheep serums examined in Adelaide.'
The causal organism, now known as Chlamydia pecorum, is still recognised as a cause of abortion in ewes and a common (perhaps ubiquitous) organism. While this organism is a well-known cause of arthritis and conjunctivitis in sheep, it was also regarded as a rare cause of abortions in ewes (Watt BR 2011, Walker et al. 2015). However, recently cases have been reported more frequently in NSW, either due to an increase in prevalence or to improved diagnostic methods. We report outbreaks of late term abortions, stillborn lambs and lambs born alive, weak and premature in ewes on five properties located in diverse environments across NSW. Foetal losses were in most case estimated at 20-30%. On Property 5, 10-12 of 130 ewes aborted before lambing but losses ceased after the ewes were injected with long acting oxytetracycline.
In each of these cases, C. pecorum was detected by quantitative PCR (qPCR) and the necropsy and pathology findings from submissions from Properties 1-3 and 5 supported the diagnosis. No other potential infectious agents were detected including Chlamydia abortus. Our findings support previous evidence that C. abortus, the cause of EAE, remains exotic to Australia.
The first outbreak (Property 1) occurred in the Mungindi district in 2018 with aborted foetuses observed from 9 September, prior to lambing, which commenced on 20 September. In all other flocks, the abortions were reported in 2020. The second case, on Property 2 occurred in 930 mature first cross ewes grazing on mixed pastures in the Pyramul district (between Bathurst and Mudgee). Lambing commenced on 26 June and stillborn lambs were observed from 7 July. On Property 3, stillborn lambs were observed from the start of lambing in mid-July in 450, 2 and 3-year-old Dorper ewes running on high quality native pastures in the Hillston district of western NSW. On Property 4, stillborn lambs were observed from 19 August, one week before lambing was due to commence in a mob of 400 Merino ewes running on native pastures in the Bullarah district (between Moree and Collarenebri). The final case (Property 5) occurred in 130 maiden Dorper cross ewes running on improved pastures on the Central Tablelands of NSW. Aborted lambs were observed from 21 September with lambing commencing on 18 October.
In addition to the abortions observed on Property 5, three lambs (one set of twins and a single) were found weak and unable to stand on 8 and 9 October prior to the (expected lambing start date 18 October). The neonates were obviously premature with a short, velvety coat and both eyes closed. The lambs were nursed with heat packs and were tube fed a colostrum/milk formula. Two of the three lambs developed the ability to suckle a bottle, but none became ambulatory despite assistance. The producer considered that they were having difficulty breathing. The lambs survived for 1-4 days.
Laboratory findings - necropsy and histopathology
Three lambs were submitted to Elizabeth Macarthur Agricultural Institute (EMAI) for necropsy from Property 1 in 2018. The findings have been described in detail (Westermann et al. in press), but in summary the foetuses were estimated to be from 112 to 126 days of gestation. Only minor gross changes were seen in the foetuses although one was covered in meconium. The two accompanying placentae had diffusely red-brown cotyledons and patches of thickened opaque or pink intercotyledonary tissue.
Samples for histopathology included two placentae and tissues from three foetuses. Lesions are summarised as follows:
Lamb 1: Severe necrosuppurative placentitis and vasculitis affected both cotyledonary and intercotyledonary areas of the chorioallantois. The kidney showed severe, multifocal, necrosuppurative pyelonephritis. Moderate, multifocal, necrotising cryptitis (inflammation of intestinal crypts) was present within the intestine. Bronchioli contained intraluminal mucinous material admixed with cellular debris, neutrophils, macrophages, meconium deposits and squames. No significant lesions were seen in the brain, liver, heart, spleen and abomasum.
Lamb 2: Placental chorionic villi were mildly hypercellular with cellular debris present but no vasculitis. The allantoamnion revealed mild, multifocal infiltrates of mixed macrophages with occasional lymphocytes and neutrophils. The brain showed mild to moderate, multifocal gliosis and mild lymphohistiocytic perivascular cuffing and leptomeningeal infiltrates. Abomasum and intestine contained mild to moderate mucosal infiltrates of lymphocytes, macrophages and fewer neutrophils. No significant lesions were seen in the liver, lung, heart, spleen.
Lamb 3: The kidney revealed multifocal desquamation of proximal tubular epithelium, which was replaced by cellular debris and low to moderate numbers of neutrophils. No significant lesions were seen in the brain, liver, lung, heart, spleen and abomasum.
On 8 July, three lambs were necropsied from Property 2. No placentae were presented. All were small (crown rump lengths 40-45 cm) but near term. The second lamb appeared 'pot-bellied,' and was meconium stained. The lungs were mottled pink and red and the liver was enlarged with rounded edges and fibrin tags on diaphragmatic surface. Intestines adhered together with yellow strands (Figure 4). On 24 July another near-term lamb from this mob was necropsied (case 4). The lungs mottled pink/red and the liver was mildly enlarged and dark red. The foetal membranes were dark red, with occasional white mottled areas.
Histological lesions for 3 lambs were as follows:
Lamb 2: Fibrinous serositis affected the liver surface, portal triads contained moderate infiltrates of macrophages and fewer lymphocytes. Myocardium and epicardium contained mild to moderate, multifocal infiltrates of macrophages, fewer lymphocytes and occasional neutrophils. No significant lesions were seen in the lung.
Lamb 3: The liver contained mild portal infiltrates of macrophages with fewer lymphocytes and plasma cells; there was also mild, multifocal, individual hepatocyte necrosis. Endocardium, myocardium and epicardium contained moderate, multifocal infiltrates of macrophages, fewer lymphocytes and plasma cells. No significant lesions were seen in the lung.
Lamb 4: The liver contained mild, multifocal infiltrates of macrophages and lymphocytes, together with some haematopoietic cells. The placenta contained moderate, multifocal infiltrates of macrophages with fewer lymphocytes and plasma cells within the chorioallantoic stroma, and there was suspected ulceration of chorionic epithelium (obscured by autolysis). No significant lesions were seen in the lung.
On 22 July, two dead lambs were submitted to EMAI for necropsy from Property 3. Both were small (1.4-1.5 kg) and well preserved with lungs that were not aerated. One lamb had fibrin tags on the liver surface and multifocal red blotches on the lung.
Foetal tissues from two lambs and placenta from one lamb were available for histopathology. Findings were as follows:
Lamb 1: Histopathology revealed fibrinocellular serositis of the liver. No significant lesions were seen in the kidney, lung, adrenal gland and brain.
Lamb 2: The chorioallantois was autolysed, however mild to moderate infiltrates of mononuclear inflammatory cells could be seen adjacent to blood vessels and within connective tissue. No significant lesions were seen in the lung, heart and kidney.
On 19 August, two dead lambs were submitted to EMAI from case 4. Both were well preserved, near normal in size (2.8 and 3.3 kg) and judged to be close to term. Both lambs had inflated lungs, but it appeared that neither had walked. One had moderate meconium staining and one had evidence of predation around the time of death. Otherwise there were no significant gross findings.
No histopathology was performed.
On 2 October, a single, small, cat-sized, pre-term foetus from Property 5 was necropsied. While some subcutaneous serosanguinous fluid was observed no gross changes were seen in the chest and abdomen. The placenta was grossly normal.
Tissues received for histopathology were moderately autolysed. No histological lesions were seen in the placenta or foetal liver, lung, heart and kidney. Autolysis may have obscured mild inflammatory lesions.
On 6 October, twin lambs were submitted to EMAI. The lambs had not walked or suckled but had breathed. Placentae were moderately autolysed, but without obvious lesions of placentitis. One lamb had slightly increased pericardial fluid volume with fibrin present. Histopathology was performed on lamb 2.
Lamb 2: The chorioallantois showed evidence of a severe necrosuppurative placentitis and vasculitis. In the liver there were mild portal infiltrates of macrophages and fewer neutrophils. No significant lesions were seen in the lung, kidney and heart.
Summary of pathology findings - necropsy and histopathology
Placentae: Six placentae were examined at necropsy from three of the five properties. Gross lesions were only described in one. It was described as having “patches of thickened opaque or pink intercotyledonary tissue”. No obvious lesions were described in the other placentae, though all five foetuses had positive Chlamydia qPCR results on placental and/or foetal tissues. Histologically two of the six placentae had lesions of severe placentitis; three showed a mild to moderate placentitis; one did not have any significant inflammatory lesions.
Foetuses: Fourteen lambs were examined by necropsy. Gross lesions were only described in two of them. Both had fibrinous exudate on the surface of the liver. One had fibrinous exudate on the diaphragmatic surface of the liver, as well as fibrinous exudate over the serosal surface of the intestines. The other had fibrin tags on the liver surface. Histologically two of nine livers had fibrinous serositis; four of nine livers had mild to moderate non-suppurative hepatitis involving mainly portal areas; one of two intestines had inflammation of intestinal crypts (cryptitis); one of three brains had mild non-suppurative encephalitis; two of six kidneys had inflammatory lesions; two of eight hearts had mild non-suppurative inflammatory infiltrates; one of 10 lungs had mild luminal inflammation and debris within bronchiolar lumina; none of three spleens had lesions.
C. pecorum was detected by qPCR in various foetal tissues from all five Properties. The highest loads (gene copies) of C. pecorum were detected in foetal membranes/placenta and abomasal contents (Table 1).
Table 1. Quantitative PCR results from tissues collected from cases 1-5.
|C. pecorum gene copies
|Abomasal fluid Lamb 1
|Abomasal fluid Lamb 2
|Abomasal fluid Lamb 3
|Liver Lamb 1
|Liver Lamb 2
|Liver Lamb 3
|Lung Lamb 1
|Lung Lamb 2
|Lung Lamb 3
|Heart Lamb 1
|Heart Lamb 2
|Heart Lamb 3
|Kidney Lamb 1
|Kidney Lamb 2
|Kidney Lamb 3
|Spleen Lamb 1
|Spleen Lamb 2
|Spleen Lamb 3
|Brain Lamb 1
|Brain Lamb 2
|Brain Lamb 3
|Foetal membrane Lamb 2
|Foetal membrane Lamb 3
|Abomasum fluid 01
|Abomasum fluid 02
|Abomasum fluid 03
|Stomach content Lamb 1
|Stomach content Lamb 2
|Liver Lamb 1
|Liver Lamb 2
|Liver and lung Lamb 1
|Liver and lung Lamb 2
In all Properties C. abortus was ruled out by qPCR. Toxoplasmosis was excluded in four of the five properties (and not tested in the fifth), either by the latex agglutination test on pleural fluid or by serological testing of ewes that were presumed to have aborted. Abomasal content samples from all five properties were negative for Campylobacter species. Pestivirus was excluded from submissions from three properties either by serological testing of ewes presumed to have aborted or by testing the pleural fluid from foetuses. Q fever was excluded by qPCR from two properties and Leptospira spp. were also excluded by qPCR in one case.
Where possible, producers were advised to collect aborted lambs and placentae and remove ewes that had aborted. In most situations, especially with large paddocks, this was not practical. Oxytetracyclines are effective against chlamydia and have been used both in C. pecorum (Williams, 2019) and in C. abortus outbreaks of abortions (Aitken and Longbottom, 2007). On Property 2, the owner decided not to use antibiotics because by the time the diagnosis had been made, lambing was well underway making mustering and treatment both impractical and of doubtful value.
On Property 5, 10-12 stillborn foetuses were observed in the two weeks before the due lambing date. With a diagnosis of chlamydial abortion, the remaining ewes were treated with long acting oxytetracyclines (Alamycin LA 300, oxytetracycline 300 mg/ml, Norbrook, 8 ml intramuscular dose) on 12 October, six days before the due lambing date of 18 October. In the week following antibiotic treatment no lambs were born but lambing commenced on the due date with the next 80 lambs being born normally apart from a small number of large lambs that died mainly associated with dystocia.
In its previous iterations as 'the psittacosis-lymphogranuloma agent responsible for enzootic abortion in ewes', the 'virus of enzootic abortion', Chlamydia psittaci, Chlamydophila pecorum and now C. pecorum, this highly successful and important pathogen of livestock and some wildlife species (most notable the koala), has been suspected or identified as an unusual cause of abortions and stillbirths in Australian sheep since the 1960's.
Hughes et al. (1964) found four suspicious diseased placentas from 3,503 dead lambs (and placentae when available), collected from properties in the Oberon, Orange and Monaro areas in 1963. In Tasmania, Mundy et al. (1966), tentatively diagnosed 'the virus of enzootic abortion,' in one of 153 outbreaks of ovine abortions investigated between 1960 and 1965. Hore et al. (1973) investigating viral as well as other pathogens in perinatal lamb mortalities from 66 properties in Victoria in 1969-70 found no evidence of the 'gross changes seen in outbreaks of enzootic abortion in ewes as occurs in Scotland,' but added 'this does not suggest that chlamydiae may not be important in ovine reproductive failure in Victoria'. Dennis (1974) after examining carcasses and placentae from lambs on 695 farms in the agricultural district of Western Australia from 1963-1965 made a 'tentative diagnosis of enzootic abortion' in one flock of 80 ewes that aborted 12 foetuses. Plant et al. (1972), summarising the findings from submissions from 368 NSW flocks between 1963 and 1970, reported that congenital infections were diagnosed on 30.4% of these flocks (with vibriosis diagnosed on 14.4% of properties, toxoplasmosis on 10.1% and listeriosis on 7.6%) but he reported no cases suggestive of chlamydial involvement. More recently, Williams (2019) published the first reported case of C. pecorum (in conjunction with Hairy Shaker Disease virus) causing abortions ('at any scale') in a large Romney flock in New Zealand.
Seaman (1985) cultured a chlamydia from two lambs stillborn two weeks prematurely from one flock, and from a placenta from an aborting ewe in another flock. He noted that one of the lambs had an enlarged liver covered with fibrinonecrotic material and that the placenta was oedematous with yellow-brown intercotyledonary exudate and necrotic foci in the cotyledons. On histological examination of the placenta he found placentitis and vasculitis and, using Giemsa stain, demonstrated elementary bodies in the chorionic epithelium. He also found inflammatory lesions in the liver, heart and lung. Seaman remarked that his findings and those of previous surveys, mentioned above, 'indicate that in contrast to many other countries the prevalence of chlamydial abortion in sheep in Australia is low, presumably the result of managemental or environmental factors or differences in host resistance and virulence.'
The detection of C. pecorum by qPCR provides strong suspicion of abortion due to C. pecorum infection but does not necessarily confirm it as the cause. Subclinical intestinal infections have also been reported in ruminants and could cause potential contamination of samples. Faecal contamination would presumably be less likely to affect foetal than placental samples, though, since qPCR is so sensitive, one should be mindful of cross-contamination when sampling. In submissions from these five properties qPCR was performed on a varying range of samples. These included placentae and foetal samples such as abomasal content, lung, liver, kidney, spleen and heart. While C. pecorum could be variously detected in all sample types, foetal membranes/placentae and abomasal contents generally contained the highest loads of C. pecorum. High loads of C. pecorum within the placenta are consistent with the observations of placentitis associated with current and historical cases of C. pecorum-associated abortion. While gross and histological changes were observed in the liver in several cases described here, the load of C. pecorum in liver tissue was relatively lower. Thus, qPCR testing of liver tissue alone in the case of Property 4 was not informative with respect to confirming diagnosis. The qPCR findings may be helpful in guiding future sample selection where C. pecorum abortion is suspected.
Histological evidence of a severe placentitis with vasculitis, though not pathognomonic, provides additional evidence for chlamydial abortion. In the five outbreaks described in this paper, histological evidence of severe placentitis was present in two of six placentae examined, though mild to moderate placentitis was present in another three placentae. When sampling the placenta for histopathology, lesions of thickening, opacity, oedema or exudate should be sought, but might not be obvious. Sampling placentae from more lambs also increases the likelihood of detecting severe placental lesions. This occurred in Property 5, where one placenta had no lesions, but a second placenta had a severe placentitis with vasculitis. Histological lesions in foetal tissues are variable and not specific but provide additional supportive evidence. The finding of inflammation affecting intestinal crypts (cryptitis) in some foetuses, suggest that small intestine may be useful as an additional sample for histopathology.
We are mindful of the comments made by Dennis (1967) who reported an outbreak of ovine abortion associated with three pathogens; 'the outbreak further emphasises the danger of examining only a few lambs when endeavouring to reach an accurate diagnosis of ovine abortion.' We acknowledge that a limited number of lambs and placentae were examined. Our testing for alternate causes of abortions, while not comprehensive, failed to find any evidence of other pathogens.
Until 2018, we also regarded C. pecorum as a rare cause of abortions in ewes in NSW (Watt 2011). However, since the case reported from Mungindi in 2018, either because of increased surveillance, increased prevalence or better diagnostic methods, we have found that C. pecorum has caused significant foetal and lamb losses in at least five flocks across NSW. If this is an upsurge rather than an aberration, the question is why, given that C. pecorum is probably near ubiquitous?