{'fraction_non_alphanumeric': 0.03615425816818425, 'fraction_numerical': 0.022495982860203535, 'mean_word_length': 4.812451361867704, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Seroprevalence detection of Coxiella burnetii antibodies in milk and serum of dairy cattle by recent methods

Seroprevalence detection of Coxiella burnetii antibodies in milk and serum of dairy cattle by recent methods Ashraf A. Abd El-Tawab, Fatma I. El-Hofy, Mona M. Sobhy, Rehan Tag Department of Bacteriology, Immunology & Mycology, Faculty of Veterinary Medicine, Benha University, Egypt. Department of Reproductive Diseases. Animal Reproduction Research Institute ARC, Giza, Egypt Organization of Veterinary Services, Dokki, Giza, Egypt


INTRODUCTION
Q fever is a zoonotic and prevalent disease in most countries of the world, it caused by gram negative bacteria called Coxiella burnetii. This organism has many hosts include mammals, birds and arthropods like ticks (Angelakis and Raoult, 2010). Coxiella burnetiia Gam-negative intracellular bacterial pathogen is the causative agent of Q fevera widespread zoonotic disease (Boroduske, et al., 2017). Dairy cattle have been the main source of human infections during latest outbreaks of Q fever (Wallensten, et al., 2010). Q fever has been recognized since 1952 when C. burnetii was first isolated from ticks collected at the Cairo Abattoir on infested camels and bulls imported from Sudan (Taylor, et al., 1952). The shedding of C. burnetii into the environment happens primarily by birth products, especially placenta, are heavily infected with C. burnetii also gained from milk and other excreta of infected animals (Schmeer et al., 1987). Coxiella can persist in the environment for long periods and carried for a long distance through the wind (Kirkan et al., 2008). In dairy cattle, late abortion, mastitis and infertility are the major clinical symptoms related to Q fever (Arricau et al., 2003). Q fever can also be spread by ticks which pass the bacteria from an infected to a susceptible animal, and whose feces contain the bacteria thus also contaminating the environment. Since it is also shed in the milk of an infected animal, it can be contracted by drinking unpasteurized infected milk (OIE, 2018) Animal birth products either from abortions or normal deliveries were the highest risk for animal and human infections due to a high load of C. burnetii in placental tissues. Aerosols containing airborne particles from dairy farms, especially animal birthing places and farms with high cow's abortion rates are considered to be the highest risk for human infection (Kersh et al., 2013). Increased risk of C. burnetii infections in animals associated with local many factors such as regional herd density, herd size, animal housing system, animal movements, hygiene practice as well as climatic and geographical characteristics of the region (Nusinovici et al., 2015). Molecular genotyping data does not exclude cattle as a possible reservoir of C. burnetii strains infectious to humans (Roest et al., 2013). The seroprevalence is associated with cattle farming has revealed seropositive individuals, suggesting that cattle-human contact can be a common route of infection (Schimmer et al., 2014

Samples:
Milk and serum samples were collected from apparently healthy cows from farms (n=300) consisted of pooled milk and serum (n= 150) from Giza, Fayoum and Beni Suief Governorates. Serum samples were collected in Vacutainer tubes under aseptic conditions from dairy farms cows. Pooled milk and sera samples were transferred in sterile screw capped bottles and stored at -20º C until processed.

Indirect fluorescent technique:
The detection of IgM and IgG antibodies in serum and milk was done using commercial Kit's manufacturer VIRCELL*, (SPAIN) according to Soriano et al. (1993).

ELISA test:
Commercial ELISA test kits (IDEXX Laboratories, USA) were used to detect anti-C. burnetii IgG antibodies. The sample optical densities (OD) were measured by a microplate ELISA reader (Biomed, USA) at 450 nm according to Schelling et al. (2003).

Rt-PCR taq Man:
DNA from milk or serum samples was extracted using the Roche kit (High Pure PCR Template Preparation Kit) according to the manufacturer's instructions. An assay based on the use of Taq-Man probe was performed to screen the samples. Isolated DNA samples were tested using primers and a probe targeting the repetitive transposon like region targeting CB IS1111 element were performed of the C. burnetii genome (Hoover et al. 1992).  (Table 4). ELISA test gives specificity 100% and sensitivity 98% more than other serological tests used to diagnose coxiellosis (Table 6). Fig. 1 The green fluorescence of IgM to C. burnetii phase II under ultraviolet light at a magnification of 400 X by fluorescence microscope Fig. 2 The apple-green fluorescence of IgG to C. burnetii phase II under ultraviolet light at a magnification of 400 X by fluorescence microscope

DISCUSSION
First report of Q-fever outbreak was recorded in Australia in 1935 (Setiyono et al., 2005) and a zoonosis reported worldwide (Greenslade, et al.2003), C. burnetii is the causative agent, has a wide range of animal reservoirs (Li, et al., 2005). Diagnosis of Q fever is based on the detection of specific antibodies produced against the organism following exposure (Dupont, et al., 1994). Serological tests have proven to be inadequate for detecting Q fever infections in the very early phase of the disease, when antibody levels are low or developing (Carrillo, et al.2009). The indirect immunofluorescence assay (IFA) is now considered the reference method or "Gold standard" for the diagnosis of Q fever by serology, the most sensitive and specific for the detection of Coxiella antibodies (Herremans, et al. 2013).
In the current study, IFAT revealed the presence of IgM antibodies for C. burnetii where the seroprevalence of C. burnetii were 6% of cow dairy milk, 7.3% of sera, IgG antibodies 10.6% of milk and 24% of dairy cow sera. The highest infection was recorded in Beni Suief 8% for IgM antibodies in both milk and sera. But the highest infection was recorded in Giza 30% for IgG antibodies in sera and 12% in dairy milk of Beni Suief. In this respect, Setiyono et al., (2005) reported that IFA results are the most specific and sensitive for phase II and phase I IgG antibodies and, to a lesser extent, also for the phase II and phase I IgM antibodies in order to determine which animal represents a current risk for transmission, as animals may shedding or remain seropositive long after the acute infection. The enzyme linked immunosorbant assay (ELISA) designed to measure antibody levels which have high sensitivity and specificity. This assay can be automated and reduce turnaround times and produces a measurable end point which allows for standardization of the assay results (Williams, et al. 1984). In the current study, positive samples by ELISA test were 25.7%; in serum 34% and in milk 17.3% respectively. The highest infection by ELISA test was in Beni-Suief 28% followed by Giza 25%. These results agreed with the previous results reported by Parker, et al. (2006). ELISA test gave specificity 100% and sensitivity 98% (Waag, 1995

CONCLUSION
We concluded that dairy cows should be the focus of any further research to establish their role in the transmission of C. burnetii to humans and to identify any potential risk factors for exposure. Using PCR as well as serological surveys of animals are important methods for diagnosis and control of Q-fever. Awareness rising is needed for animal owners, veterinarians, physicians and authorities.