Distinguishing between ovine abortion and ovine arthritis Chlamydia psittaci isolates with specific monoclonal antibodies

Monoclonal antibodies to Chlamydia psittaci were prepared by both in vivo and in vitro immunization methods, using an abortion strain of C psittaci as the immunizing antigen. Seven of the 8 monoclonal antibodies produced were genus-specific by the enzyme-linked immunosorbent assay and immunofluorescence test. The genus-specific antibodies were reactive with a protease-resistant, periodate-sensitive antigen of less than 14 kilodaltons. The remaining monoclonal antibody, 10D7, was specific for ovine abortion strains of C psittaci and nonreactive with 2 strains isolated from the joints of lambs with polyarthritis. The type-specific antigen was protease sensitive, but could not be detected in the immunoblot assay.     

Comparison of some ovine Chlamydia psittaci isolates by indirect immunofluorescence

Four ovine abortion isolates, including the A22 vaccine isolate, and an isolate from a case of lamb arthritis, were compared by indirect immunofluorescence using antisera raised in chickens and mice. Cross titrations with homologous and heterologous antisera showed a one-way reaction between the single chlamydial isolate from a lamb with arthritis and the four isolates from cases of ovine enzootic abortion. The abortion isolates could not be distinguished.     

Antibody response of the ovine lymph node to experimental infection with an ovine abortion strain of Chlamydia psittaci

After primary infection with Chlamydia psittaci in the draining area of the popliteal lymph node, viable organisms could be isolated from the efferent lymph only before the primary immune response developed. The lymph antibody response, as assayed by the complement fixation and immunofluorescence (IF) antibody tests, showed a rise in titre that peaked approximately 2 weeks after infection. Immunoblotting revealed that antibodies produced during this period were predominantly directed against the major outer membrane protein (MOMP). In secondary infection of convalescent sheep, an elevated IF antibody titre, already present in the lymph and blood, could not be boosted. Viable organisms could not be isolated from these sheep. Antibodies produced reacted to 12-14 bands in the immunoblot profile including the MOMP band. These potentially immunoprotective antigens, particularly MOMP, should be considered as useful candidates for an improved vaccine against ovine enzootic abortion, in further investigations.     

Detection of Chlamydophila abortus in ovine milk by immunomagnetic separation-polymerase chain reaction

The present study was carried out to investigate the presence of Chlamydophila abortus, the causative agent of ovine enzootic abortion, in milk samples collected from sheep flocks with and without the history of abortion in Eastern Turkey by means of immunomagnetic separation (IMS) in conjunction with the polymerase chain reaction (PCR). A total number of 201 milk samples collected from 10 flocks with abortion and four flocks without abortion were tested. In the analysis of the milk samples by IMS-PCR, correct amplification was obtained with three (1.5%) samples originating from one flock with abortion. In the digestion of PCR positive products by AluI, restriction profiles observed in all three samples were determined to be the same as C. abortus S26/3.     

Use of the IDEIA ELISA to detect Chlamydia psittaci (ovis) in material from aborted fetal membranes and milk from ewes affected by ovine enzootic abortion

The IDEIA ELISA was used to detect Chlamydia psittaci (ovis) antigen in ewes' milk to which were added serial dilutions of chlamydiae titrated as inclusion forming units (ifus) in McCoy cell tissue culture. The test was able to detect as few as 35 ifus/ml of the organism. The ELISA was then used to detect chlamydial antigen in fetal membranes and milk from ewes clinically affected with ovine enzootic abortion (OEA). The results were compared with results of isolation of chlamydiae in McCoy cell tissue culture from the same material. The fetal membranes of 17 of 19 ewes were positive for chlamydia when tested with the ELISA but chlamydia could be cultured from only 15 of them. Milk samples from 26 ewes which had aborted between 1 and 34 days previously were tested: chlamydiae could not be cultured from any of them and only one was positive when tested by the ELISA. The results show that the IDEIA ELISA is a sensitive test for the detection of C. psittaci (ovis) antigens. The positive results to this test for the three samples from which chlamydiae could not be cultured suggest that the test is not as specific as culture or that it detected dead organisms. Chlamydiae do not appear to be excreted in the milk of ewes affected with OEA.     

Comparison of protein patterns between invasive and non-invasive ovine strains of Chlamydia psittaci

Protein patterns displayed in sodium dodecyl sulphate (SDS) electrophoresis by invasive and noninvasive strains of Chlamydia psittaci showed three constant differences, the most distinctive being a band at 90 Kd from invasive strains. In comparison with other methods so far described, this method provides a more efficient means of differentiating between invasive and non-invasive strains. Furthermore, it may lead to the development of improved methods of diagnosis.     

Chlamydia psittaci: is tonsillar tissue the portal of entry in ovine enzootic abortion?

Ewes at 70 days of gestation were exposed by various routes to a culture of Chlamydia psittaci. Chlamydial infection of fetal tissues, generally accompanied by abortion, was observed only in four of six ewes inoculated via the tonsillar crypts and in five of seven ewes injected subcutaneously; most of these also developed antibodies to C psittaci. Seroconversion after normal lambing was also observed in most ewes inoculated orally or by stomach tube, despite failure to detect chlamydia in them.     

Usefulness of subjective ovine milk scores: II. Genetic parameter estimates

Genetic parameters for a subjective milk score given to ewes within 24 h of parturition were estimated to determine the usefulness of milk score as a selection trait to improve milk production, which influences total litter weight weaned. Heritability of milk score and the genetic correlation of milk score with litter weight weaned were estimated by REML separately for four sheep breeds, Rambouillet (n = 1,731), Targhee (n = 1,638), Columbia (n = 1,731), and Polypay (n = 1,129). Litter weight weaned was the total weight of lambs weaned at approximately 120 d of age under a western range production system. Observed heritability estimates for milk score at first parity were moderate and similar among breeds, ranging from 0.18 to 0.32. Heritability estimates adjusted for a binomial distribution of milk scores at first parity were high (Columbia, 0.43; Polypay, 0.35; Rambouillet, 0.50; Targhee, 0.84). Estimates of observed heritability for second-parity milk score were moderate to high, ranging from 0.23 to 0.46. Milk score at first or second parity was genetically correlated with milk score records at maturity (third parity and greater), with estimates ranging from 0.69 to 1.00. Milk score and litter weight weaned were genetically correlated at first or second parity in Rambouillet (r(g) = 1.00) and Targhee breeds (r(g) = 1.00 and 0.61, respectively), but not in the Columbia and Polypay breeds. Estimates of heritability for lifetime records for milk score ranged from 0.16 to 0.26 across breeds. Estimates of genetic correlations of annual lifetime milk score records with litter weight weaned were high (Columbia, 1.00; Polypay, 0.81; Rambouillet, 1.00; and Targhee, 0.77). Repeatability estimates for milk score were similar across breeds, 0.23 for Columbia, Rambouillet, and Targhee ewes and 0.28 for Polypay ewes. Milk score measured at first or second parity may be a good predictor of future potential milking ability. Further, milk score can be used as a selection trait to improve maternal ability for increasing litter weight weaned. The need for increasing ewe milking performance and lamb growth rate at first parity in commercial range sheep production systems may be addressed by selection for milk score at first parity.     

Antigenic and morphological differentiation of placental and intestinal isolates of Chlamydia psittaci of ovine origin.

Ewe placental and lamb intestinal isolates of Chlamydia psittaci recovered from flocks affected with ovine enzootic abortion were examined by inclusion morphology, indirect immunofluorescence (IIF) and immunoblot analysis. Chlamydiae recovered from the faeces of sheep from two flocks free of clinical disease were also examined. In cell culture ovine abortion (OA) and intestinal isolates were distinguishable by inclusion development and morphology. Similarly, in two-way IIF tests with one week mouse antisera isolates fell into two distinct groups: abortion or intestinal. Immunoblotting with convalescent sheep abortion antiserum identified 30 out of at least 40 silver staining polypeptides as antigenic both in OA and intestinal isolates. The serum produced a similar reaction pattern to the resolved proteins of each OA isolate, indicating a higher degree of antigenic conservation among these isolates. Considerable cross reactivity between the OA and intestinal isolates was identified, but the serum also showed apparent molecular weight differences between antigens of the two types in the 87-116 kDa, 38-44 kDa and 26-28 kDa regions. Furthermore, the immunoblotting analysis revealed heterogeneity among the intestinal isolates, particularly in antigens between 87-116 kDa and 38-44 kDa.     

Efficacy against ovine enzootic abortion of an experimental vaccine containing purified elementary bodies of Chlamydia psittaci

A vaccine prepared from purified, inactivated elementary bodies of Chlamydia psittaci protected sheep against abortion after subcutaneous challenge with live chlamydiae. Immunoblot analysis of serum samples revealed a consistently dominant antibody response against the chlamydial major outer membrane protein in all vaccinated sheep. Reactions to other chlamydial antigens were also detected but were less pronounced or inconsistent. Serological responses detected by complement fixation were variable and did not correlate with immunity.     

Pathology in the ovine foetus caused by an ovine pestivirus

Homogenised tissues or tissue culture supernatant fluid containing a noncytopathic pestivirus obtained from a lamb with a neurologic form of border disease, were inoculated into ewes at different stages of pregnancy. Foetal death occurred in 9 ewes of those inoculated between 19 and 47 days of pregnancy while 3 ewes did not lamb. Eight of the foetuses were aborted between 77 and 132 days of pregnancy; of these 6 were autolysed or mummified and one had arthrogryposis. The one full-term dead lamb had a hairy birth coat and lissencephalic micrencephaly. Foetal death occurred in only 7 of 14 ewes inoculated between 57 and 72 days of pregnancy. Four of these ewes aborted between 77 and 108 days of pregnancy and 3 gave birth to full-term, dead, hairy lambs. The remaining 7 ewes gave birth to live hairy lambs with severe inco-ordination. All lambs carried to term and aborted foetuses or lambs that could be examined had a range of intracranial malformations including focal leucomalacia, micrencephaly, hydranencephaly, porencephaly, lissencephaly and cerebellar hypoplasia. Some lambs also had skeletal abnormalities including arthrogryposis, scoliosis and brachygnathia inferior. The pestivirus isolate used in these trials produced more severe effects on the ovine foetus than previously observed in similar inoculation trials using pestivirus isolates from border disease lambs without nervous signs.     

Usefulness of subjective ovine milk scores: I. Associations with range ewe characteristics and lamb production

Range ewes are commonly evaluated for milking ability by producers to determine the ewe's ability to rear lamb(s). The U.S. Sheep Experiment Station has subjectively scored (low, average, high) a ewe's milking ability within 24 h of lambing for many years. The relationship of subjective milk scores with lamb production was investigated using lambing records of Columbia (n = 1,731), Polypay (n = 1,129), Rambouillet (n = 1,704), and Targhee (n = 1,638) ewes. The incidence of high milk scores increased from less than 10% at first parity to 29 to 40% at second and greater parities. At maturity, Columbia ewes (38%) had the highest percentage of high milk scores. A positive association existed between ewe BW and her milk score at third and later parities. Ewes with high milk scores gave birth to heavier lambs (P < 0.05), whereas ewes with low milk scores were associated with lighter (P < 0.05) lambs at birth. Ewes with low milk scores weaned less (P < 0.05) total weight than ewes with better milk scores across all age groups for all breeds. Lighter weaned litter weights from ewes with low milk scores were linked to lighter birth weights and fewer weaned lambs. Differences for litter weight weaned between ewes with average and high milk scores were generally observed at 2 and 3 yr of age, when litter weights were heavier among ewes with high milk scores (P < 0.05) for all breeds. Between the ages of 1 and 3 yr, Columbia, Polypay, Rambouillet, and Targhee ewes with an average milk score weaned heavier (P < 0.05) litters (average differences of 10, 9, 13, and 12%, respectively) than ewes with low milk scores. For all breeds at all ages, individual lamb weaning weights were heavier (P < 0.05) when they were reared by ewes with high milk scores compared to lambs reared by ewes with low milk scores. Results suggest that milk score is an economically important trait in these four breeds and should be considered in management and breeding objectives; at a minimum, the incidence of low milk scores should be kept as small as possible.     

Effect of farm characteristics and practices on hygienic quality of ovine raw milk used for artisan cheese production in central Italy

A survey on ovine dairy farms directly transforming own‐produced milk, in the Italian Marche region, was carried out to assess flock and milking practices that may influence milk hygienic‐sanitary conditions. A census survey established that 24 dairy farms were located in this region. Bulk milk samples were collected throughout the milking period in each dairy farm in 2013. Analyzed variables were: (i) chemical parameters such as fat, protein and lactose content, dry matter and pH; and (ii) total bacterial (TBC) and somatic cell counts (SCC). Chemical parameter values were in agreement with published data while, geometric mean (GM) log₁₀ SCC was 5.91 and TBC GM was 57 978 colony forming units/mL, in compliance with Eropean Union criteria. A positive correlation was found between SCC and TBC when GMs of all farm data were considered (Spearman's rho = 0.7925; P = 0.0001). Statistical analysis did not show significant correlation between SCC or TBC GM and dairy farm principal characteristics. Although SCC levels detected in the present study should suggest the need to implement mastitis control programs, Marche's dairy sheep flocks revealed a good hygienic condition level. This is an important aspect in implementing safety for end users of the final product.     

Comparison of isolates of Chlamydia psittaci of ovine, avian and feline origin by analysis of polypeptide profiles from purified elementary bodies

The single species Chlamydia psittaci is a diverse grouping which contains several different types of chlamydial strain for which there is no generally accepted typing method. The results obtained when profiles of polypeptides from purified elementary bodies are compared are consistent with type designations obtained using other criteria. However, the method still requires large scale culture and extensive purification of the chlamydial cells.     

Coagulase negative staphylococci from ovine bulk-tank milk: Effects of the exposure to sub-inhibitory concentrations of disinfectants for teat-dipping

Teat-dipping is one of the most effective methods to prevent mammary infections in ruminants, including sub-clinical mastitis caused by coagulase-negative staphylococci (CoNS). Improper disinfectant application could expose microorganisms to sub-inhibitory concentrations leading to phenotypic variations. In this study, 12 chlorhexidine-digluconate (CHDG)-tolerant (of which 4 qac positive) and 12 benzalkonium chloride (BC)-tolerant (of which 7 qac-positive) CoNS isolates from ovine milk were exposed to sub-inhibitory concentrations of CHDG and BC, respectively. Changes in disinfectant susceptibility against BC and CHDG, antibiotic resistance against 12 antibiotics and biofilm production were then assessed for both groups. After CHDG stress, 67 % and 83 % of the CHDG-stressed isolates doubled their MICs for BC and CHDG, respectively and 2 qac-negative isolates showed a four-fold increase of their MBCs for CHDG. After BC stress, MICs for BC and CHDG doubled in 58 % and 83 % of the BC-stressed isolates, respectively, while one qac-positive isolate increased four-fold the MIC for BC. Cross-resistance to antibiotics was assessed by disc diffusion method. Some qac-positive isolates varied their resistance profile, while a blaZ-positive isolate showed a resistant phenotype against ampicillin only after the exposure to the disinfectant. As for qac-positive isolates, one CHDG-stressed and 2 BC-stressed increased their resistance to kanamycin and cefoxitin, respectively. The Congo Red Agar test was carried out to assess the in vitro slime production: all isolates were negative after stress. In conclusion, sub-inhibitory exposure to disinfectants may affect disinfectant and antibiotic susceptibility, the latter in particular for qac-positive isolates and those hosting unexpressed antibiotic resistance genes.