Sensitivity and specificity of some serological techniques by type of antigen, and evaluated population
Visceral leishmaniasis (VL) is a serious public health problem of great medical and veterinary importance. This disease is endemic in Brazil and in many other countries of Latin America, Asia, Africa and Europe (1). According to recent review (2), approximately 0.2 to 0.4 million cases of VL occur each year and although worldwide distributed, higher prevalence of the disease is concentrated in six countries, including India, Bangladesh, Sudan, South Sudan, Ethiopia and Brazil, that undertake for more than 90% of the cases. The clinical importance of VL resides in the severity of the disease that results in death of unrecognized cases and even for individuals with treatment access, death occurs in 10 to 20% of the cases [2-8].
Most of the VL cases are caused by the
The notion that dogs are the main urban domestic reservoir for this
Control strategies include performing accurate and early diagnosis of CVL to identify infected animals [19, 20]. CVL diagnosis is a difficult task since clinical signs of the disease in dogs can be confused with other diseases . In endemic areas, a large percentage of infected animals are asymptomatic or present low number of discrete signs. The role these animals play in parasite transmission is still largely unknown. Several diagnostic strategies have been implemented based on parasitological, serological or molecular methods in association with clinical and epidemiological parameters . Parasite culturing has been considered as gold standard for disease diagnosis [22, 23]. Although offering a high specificity since allows parasite identification, it offers very low sensitivity, besides it is laborious, time-consuming and largely dependent on the expertise of the observer [24, 25].
Serological tests are the most common diagnostic method employed for CVL diagnosis . Several serological methods have been implemented for diagnosis of CVL, including direct agglutination assay (DAT), enzyme linked immunoassay (ELISA) and indirect immunofluorescent antibody test (IFI) . However, most of these classical serological tests present important limitations for CVL diagnosis, including high consumption of time, and lack of sensitivity and specificity, mainly when animals present low antibody titers. This causes underestimation of disease, reflecting in failures in control measures, as well as the maintenance of infected untreated dogs in endemic areas [27, 28]. New methods based on immunochromatography have been implemented for serodiagnosis of CVL and have shown excellent results . These techniques offer several advantages since they are rapid tests easily performed even in field areas, and more specific since they use recombinant DNA technology that additionally facilitates reproducibility and large-scale production. These advantages result in better identification of infected dogs. However, the efficacy of immunochromatographic techniques for CVL diagnosis needs to be improved . In Brazil, a rapid test based in dual path platform (TR DPP®LVC - Biomanguinhos) had been recently implemented as screening test for CVL. This technique seems to be adequate to disease diagnosis in public health system. However, the TR DPP®LVC has shown an excellent performance identifying 98% of symptomatic dogs, it showed less efficacy for diagnosis of asymptomatic dogs (47%) . Since there is evidence that asymptomatic dogs can participate in natural transmission cycle of VL, new strategies should be implemented in order to improve CVL diagnosis [16, 32-34]. For serological diagnosis one strategy can be the development of rapid tests based on impregnation of multi-antigen that would offer more sensitivity, as well specificity.
Finally, it would be important to include more specific confirmatory tests for control strategies that can be advantageous to diagnose inconclusive cases. There is evidence that molecular diagnosis of
Since the discovery of canine visceral leishmaniasis (CVL) in Tunisia by Nicolle & Comte (1908), several reports have shown that dog and man share a common etiologic agent. The notion that dog is the main reservoir of visceral leishmaniasis (VL) in urban centers  is supported by several evidences including the high cutaneous parasitism observed in dogs infected by
Some studies have shown a correlation between the presence of clinical signs in infected animals and transmissibility of the parasite to the vector and, consequently, a correlation with the occurrence of human cases [16, 32, 51]. In accordance with these studies, Travi et al. (2001) and Verçosa et al. (2008) showed that asymptomatic dogs did not transmit the parasite to the vector [38, 51]. There is not a consensus about this idea, since there is a wide variation in the rates of infectivity (70 to 90%) between asymptomatic and symptomatic dogs. Studies show that, regardless of the clinical presentation, any dog has the ability to transmit
The diagnosis of VL in the dog must consider the association between clinical, laboratory and epidemiological data. As discussed above, clinical diagnosis is problematic and difficult for veterinarians to perform due to the great variability of clinical signs that
There are several laboratorial diagnosis methods for leishmaniasis: i) parasitological methods (detection of the parasite), ii) serological methods (detection of anti-
In spite of serological techniques such as enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFAT) being the most widely used methods for the diagnosis of CVL  parasitological methods, such as direct examination of slides and isolation from tissue cultures, allow the parasite to be detected and can be used as confirmatory diagnostic methods for CVL . In recent decades, molecular techniques such as polymerase chain reaction (PCR) have been introduced for the diagnosis of CVL, exhibiting high sensitivity and specificity . These techniques detect the genetic material of the parasite, which can be used as confirmatory methods in cases of recently infected or asymptomatic animals, which tend not to be diagnosed serologically, and in most cases, do not show seroconversion, having a low parasite load [4, 60]. In a study conducted in Belo Horizonte-MG, a VL-endemic area in Brazil, among 1,443 dogs evaluated, 15.3% of them were seropositive, while 84.7% showed negative serology. Interestingly, among serologically negative dogs, 24.4% showed up as positive using the molecular diagnostic technique, and most of these (97.6%) would not be diagnosed, since they consist of asymptomatic dogs with negative serology .
Dogs from endemic areas considered resistant remain clinically normal and asymptomatic without exhibiting clinical signs. There is evidence that the parasites in these animals are effectively eliminated at the infection site [62, 63]. However, in susceptible animals, a large number of parasites are detected in infected tissues. In these animals, the presence of the parasite may occur in multiple organs, accompanied by a granulomatous inflammatory reaction and production of immune-mediated phenomena, probably responsible for the appearance of various types of clinical signs .
Initial clinical signs of CVL include: hypertrophy of the lymph nodes, changes in skin appendages such as onychogryphosis, swelling of the footpad, localized alopecia, skin ulcers and nasal and periocular dermatitis. Alopecia and non-pruritic exfoliative dermatitis can spread to other parts of the animal\'s body. Weight loss may also be present, as well as cachexia, anorexia and conjunctivitis. Internal organs such as spleen, liver, kidney and lymph nodes may also be affected, when kidney injuries are present may lead to the dogs death [13, 65]. Fever, apathy, diarrhea, epistaxis, intestinal bleeding, hepatosplenomegaly, hyperkeratosis, keratoconjunctivitis are also found in affected animals [66-68]. Some clinical signs are more frequent than others; skin lesions are the most frequent manifestations affecting approximately 50 to 90% of symptomatic dogs [4, 67, 69, 70], including non-pruritic exfoliative dermatitis, with or without alopecia, which can be generalized or localized to the muzzle, ears and limbs [67, 71, 72]. Other very common signs are weight loss, observed in 25 to 80% of CVL cases, including onychogryphosis in 30 to 75%, and ocular abnormalities in 16 to 24% . The most common clinical signs of VL in dogs are depicted in Figure 1.
In dogs with CVL, clinical-pathological changes may occur such as intestinal lesions, renal and hepatic abnormalities . The main biochemical laboratory findings from CVL are hyperglobulinemia, mainly due to increased production of antibodies, and hypoalbuminemia, attributed to chronic inflammation, as long as renal and hepatic failure . The result of these changes is a reduction in the albumin/globulin ratio and hyperproteinemia . Additionally, severe CVL is associated with changes in hematological parameters such as severe anemia and leukopenia, associated with lymphopenia, eosinopenia and monocytopenia [66, 74, 75]. Immune-mediated thrombocytopenia also occurs accounting for episodes of bleeding such as epistaxis, hematuria and hemorrhagic diarrhea .
Finally, nonspecific signs of illness that are mistaken for other diseases such as babesiosis, ehrlichiosis and canine trypanosomiasis also contribute to make CVL clinical diagnosis imprecise and difficult to perform .
The detection by optical microscopy of the parasite by direct observation of stained smears from spleen aspirate, lymph node and bone marrow tissues has high specificity, allowing confirmation of CVL diagnosis [3, 53, 61, 77]. However, the sensitivity of this method is less than 30%, since the direct parasite identification may be limited, especially in mildly and asymptomatic dogs that have low parasitic load, producing false negative results [3, 53, 61, 77].
Another method that can identify the parasite in tissues is the culturing of tissue fragments or aspirates, preferably in a biphasic medium , composed by Novy-MacNeal-Nicolle (NNN), or Tobie modified medium or United States Army Medical Research Units (USAMRU) as solid phase medium and, most often, Schneider as liquid phase medium. This parasitological diagnostic method offers high specificity allowing isolation and characterization of parasites, as well as determination of which species and/or variants are circulating in endemic areas . However, the culturing consists of an indirect test, because when the parasites are isolated from various tissues, they are present in amastigote form and during cultivation they transform into the promastigote form. This process may be impaired as a result of parasite death due to a failure of temperature-control during transport of the tissue sample, or contamination during collection or cultivation . Additionally, a culturing is time consuming and may take up to 4 weeks of observation for definitive diagnosis [13, 79]. Furthermore, specific media for promastigote isolation are not easily obtained, being a technique restricted to specialized laboratories [70, 80], in which the outcome also depends on the experience of the observer [24, 25]. Although culturing offers greater sensitivity compared to direct viewing of amastigotes in tissue , it still remains at very low levels.
In summary, parasitological techniques have high specificity but low sensitivity, especially for the detection of dogs, recently infected, asymptomatic or those presenting low parasite load. In addition, the need for skilled personnel and the long delays to obtain the results prevent parasitological techniques to be used in epidemiological surveys [4, 23, 61, 81-84].
Serological tests are based on the presence of specific humoral immune responses against the pathogen or purified fraction or recombinant proteins of the pathogen. These tests allow detection of immunoglobulin (IgG) levels, thus becoming an essential tool for the diagnosis of CVL. These methods are simple to carry out and therefore they are frequently used to determine the prevalence of leishmaniasis in epidemiological studies .
A wide variety of serological methods are available for CVL diagnosis, presenting variations in sensitivity and specificity. The performance of these diagnostic techniques varies depending on the type of antigen used and the detection of anti-
The most commonly employed serological tests for the diagnosis of CVL, including ELISA, indirect immunofluorescence test (IFAT), and direct agglutination test (DAT), uses parasite or crude extract of
Despite the practicality and simplicity of serological tests, they do not have 100% sensitivity because some dogs, especially those that are resistant or in the early stages of the disease, have negative results. Thus, the results of such tests should be evaluated carefully, always associating test results with epidemiological history, clinical state of the animal, and the result of a more specific diagnostic test . In addition, since titers of anti-
IFAT is a test in which anti-immunoglobulin antibodies labeled with fluorochromes react with parasites immobilized in a slide. IFAT is a laborious technique that presents difficulties for both standardization and interpretation of the results Therefore, detection of antigen-antibody reaction by fluorescence microscopy depends on the observer experience, compromising reproducibility of this test in different laboratories. Thus, it is not considered a simple and practical technique for evaluating a large number of canine sera . In spite of these limitations, it is still being used as a diagnostic method for mass screening of infected dogs . This method varies in its performance, with sensitivity ranging from 68 to 100% and specificity of 60 to 90% [5, 88-90].
In a study evaluating IFAT for the diagnosis of CVL, the efficacy of the test was evaluated using 254 sera from infected and uninfected dogs and sera from animals with other parasitic diseases. The authors observed low sensitivity (72%) and specificity (52%), as well as cross-reactions when sera from dogs infected with other pathologies, such as
The direct agglutination test (DAT) is an alternative method for the diagnosis of VL, first described in 1975 and adapted for the diagnosis of human and canine infection in the late 1980s [93, 94]. DAT is a method that uses whole stained promastigotes as antigen, either in suspension or freeze-dried . The advantage of this test lies in its low cost when compared with other tests . However, this test is not desirable for screening large numbers of samples, since it is a laborious procedure, due to the production process for crude
Changes to the DAT protocol have been proposed by Gómez-Ochoa
For various reasons, ELISA tests based on whole parasites or crude lysate of parasite antigens for the diagnosis of CVL do not provide satisfactory results, as follows: i) it is a laborious technique, which leads to a delay in the delivery of results and, consequently, the implementation of treatment or the removal of infected dogs from endemic areas [68, 99]; ii) leads to the appearance of cross-reactions with sera from individuals infected with other
A study using 234 domesticated dogs in an endemic area for CVL assessed the efficacy of ELISA, IFAT and DAT for the diagnosis of CVL. In this study, dogs were also parasitologically evaluated for identification of
Using sera from dogs with CVL, a comparison of an ELISA test using crude soluble antigen of
Thus, the search for tests with higher sensitivity and specificity for dogs with a variety of conditions became necessary for control of CVL, which would lead to a reduction of errors in actions taken for treatment or control. In countries that adopt culling of seropositive dogs as a control measure, low sensitivity of diagnostic tests can lead to the maintenance of dogs that transmit disease and lack of specificity can result in unnecessary culling of healthy dogs. The identification of new proteins of
Another way to overcome the obstacles of ELISA based on whole parasites or crude parasite antigen was the development of ELISA tests based on parasite fractions such as that using the parasite surface molecule, fucose-mannose ligand antigen (FML). The FML-based ELISA showed a high sensitivity, which was similar in detecting either oligosymptomatic (90%) or symptomatic (90%) dogs. Regarding specificity, ELISA using crude parasite antigen for the diagnosis of oligosymptomatic dogs was superior, achieving 100% in comparison to FML-based ELISA that was 93.3%. However, for symptomatic dogs the specificity of the FML-based ELISA showed similar results of 96.7% compared to that obtained by ELISA based on crude parasite antigen (93.3%) .
Other ELISA assays based on recombinant antigens such as rA2 from
Interestingly, the association of the recombinant proteins enhanced test performance both for detection of symptomatic and asymptomatic infected dogs. Indeed, using IFAT as the gold standard, ELISA based on the mix of rK9, rK26 and rK39 from
The combination of these findings reinforces the notion that the use of multiple antigens in diagnostic tests enhances test performance and the need to search for new antigens that may compose a diagnostic test able to better diagnose asymptomatic dogs.
New recombinant proteins are being evaluated. Faria
Another study evaluated the performance of the ELISA based on another recombinant antigens of
In summary, most studies using ELISA suggest that in comparison to tests based on crude antigen, those based on recombinant antigens improves accuracy, increasing sensitivity and specificity for the diagnosis of symptomatic dogs. Although improved, test accuracy is still low for the detection of asymptomatic animals.
Recently, rapid immunodiagnostic tests have begun to be employed as routine laboratory tests for detection of diseases such as leishmaniasis. The recombinant antigens of the parasite are impregnated onto nitrocellulose membranes and serum samples are applied in the rapid test platform. Antigens impregnated in nitrocellulose membranes are recognized by specific immunoglobulin present in the serum of infected individuals. This reaction is revealed by the interaction of protein A coupled to colloidal gold particles, with the Fc portion of the immunoglobulins associated with the recombinant antigens. The use of immunochromatographic assays as diagnostic methods has the main advantages of being rapid, completed in around 15 minutes, easy to carry out and can dispense with the need for equipment to read the results . Furthermore, these tests are easily stored, and test supplies and samples do not need to be maintained at low temperatures and can it even be performed at the place of collection. These tests are already widely used to detect HIV  and H1N1  infection. For the diagnosis of CVL and human VL, among the tested and commercially available recombinant proteins, the most widely used for composing immunochromatographic tests is the recombinant protein rK39. This protein contains repetitive sequences of 39 amino acids from a protein related to kinesin of kinetoplast from
Recently, a meta-analysis was performed in order to broadly assess the performance of rapid tests using rK39 as the antigen in the diagnosis of CVL. The combined analysis of 16 studies using rapid tests based on rK39 offered a sensitivity of 86.7% (95% CI: 76.9–92.8%) for the detection of clinical disease and 59.3% (95% CI: 37.9–77.6%) for identification of
|\n \n ||\n \n ||\n \n ||\n \n |
|\n \n ||\n \n ||\n \n ||\n \n ||\n \n ||\n \n ||\n \n |
|Harith \n ||DAT\n||44\n||6\n||176\n||98.9\n||100\n|
|Barbosa-de-Deus \n ||ELISA\n||LMS\n||188\n||1582\n||55\n||98.0\n||95.0\n|
|Scalone \n ||ELISA\n||K39\n||209\n||81\n||62\n||97.1\n||98.8\n|
|Schallig \n ||DAT\n||79\n||67\n||24\n||88.6\n||96.7\n|
|Rosati \n ||ELISA\n||K26\n||202\n||20\n||0\n||100\n||100\n|
|Mohebali, \n ||Dipstick\n||rK39\n||268*\n||0\n||0\n||70.9\n||84.9\n|
|Boarino \n ||ELISA\n||K9-K39-K26 chimera\n||232\n||362\n||0\n||95.8\n||99.1\n|
|Mettler \n ||Rapid test\n||rK39\n||47\n||50\n||26\n||A: 52.9 \n |
|IFAT\n||47\n||50\n||26\n||A: 29.4\n |
|Lira \n ||EIE® - LVC\n||25\n||16\n||11\n||72.0\n||87.5\n|
|IFI® - LVC\n||25\n||16\n||11\n||68.0\n||87.5\n|
|Ferreira \n ||EIE® - LVC\n||234*\n||20\n||20\n||96.0\n||100\n|
|IFI® - LVC\n||234*\n||20\n||20\n||72.0\n||100\n|
|Ferroglio \n ||SNAP® CLATK\n||CTA\n||59\n||124\n||0\n||91.1\n||99.0\n|
|Porrozzi \n ||ELISA\n||rK26\n||100\n||25\n||14\n||A: 66.0 \n |
|ELISA\n||rK39\n||100\n||25\n||14\n||A: 66.0 \n |
|ELISA\n||rA2\n||100\n||25\n||14\n||A: 88.0 \n |
|ELISA\n||CTA\n||100\n||25\n||14\n||A: 30.0 \n |
|Cândido \n ||ELISA\n||CTA\n||60\n||30\n||0\n||O: 86.7 \n |
|O: 100\n |
|ELISA\n||FML\n||60\n||30\n||0\n||O: 90.0 \n |
|O: 93.3 \n |
|Lemos \n ||RDTs\n||rK39\n||76\n||33\n||0\n||83\n||100\n|
|Babakhan \n ||FAST\n||73\n||74\n||0\n||98.6\n||78.7\n|
|Coelho \n ||ELISA\n||LRP\n||111\n||47\n||14\n||100\n||98.2\n|
|Troncarelli \n ||IFAT\n||51\n||0\n||0\n||83.0\n||92.5\n|
|Figueiredo \n ||EIE® - LVC\n||305*\n||0\n||0\n||100\n||96.6\n|
|IFI® - LVC\n||305*\n||0\n||0\n||22.2\n||97.0\n|
|de Lima \n ||ELISA\n||CTA\n||52\n||52\n||0\n||91.5\n||94.7\n|
|Marcondes \n ||SNAP® CLATK\n||CTA\n||283\n||86\n||31\n||94.7\n||90.6\n|
|Alves \n ||EIE® - LVC\n||39\n||39\n||39\n||100\n||68.0\n|
|IFI® - LVC\n||39\n||39\n||39\n||100\n||70.5\n|
|DPP® - LVC\n||rK28\n||39\n||39\n||39\n||100\n||97.5\n|
|Grimaldi \n ||DPP® - LVC\n||rK28\n||120\n||59\n||11\n||A: 47.0\n |
|Souza \n ||ELISA\n||rLci1A\n||138\n||119\n||86\n||96.0\n||92.0\n|
|Barral-Veloso \n ||ELISA\n||31\n||37\n||45\n||93.5\n||97.6\n|
|Quinnell \n ||RDTs\n||rK39\n||322\n||59\n||0\n||46.0\n||98.7\n|
Efforts have been made to improve the efficacy of rapid tests by developing more sensitive and specific method that could be used in mass screening for the diagnosis of CVL. An alternative proposal is to use a mixture of recombinant proteins or chimeric proteins. The protein rK28 chimeric for the relevant epitopes of three antigens, rK9, rK26 and rK39 [87, 108] that showed promising efficient results in an ELISA based test , was recently used to compose a new rapid test in DPP format. This format consists of a double track platform that offers greater sensitivity and specificity . In addition, this rapid test has advantages over previously used serological methods due to greater precision, simplified interpretation of the data, minimal use of sample volumes, and compatibility with different types of body fluids such as blood, serum, saliva, plasma and urine. In contrast to these advantages, recently Grimaldi et al (2012) showed that rK28-based DPP despite its high sensitivity (98%) and specificity (96%) towards sera from symptomatic dogs, showed low sensitivity of only 47% towards sera from dogs with no signs . With regard to sera from dogs with other diseases, the observed specificity was 96%, with false-positive reactions mainly for some sera of dogs infected with
In recent decades, due to advances in molecular biology techniques and reduced implementation costs, the polymerase chain reaction (PCR) began to be used in VL diagnosis [23, 126]. Its use has demonstrated superior results to those obtained by ELISA, IFA and culture in detecting animals infected with
PCR is a technique based on the principle of complementary bases pairing of the DNA molecule, allowing amplification and detection of a particular region of the target genome using a pair of specific oligonucleotide primers. The reaction can produce tens of billions of DNA fragments from a single molecule, and has high sensitivity small quantities of samples to be used. This type of PCR, hereafter referred as "conventional PCR" (cPCR) needs electrophoresis in agarose or polyacrylamide gels along with dyes such as ethidium bromide, SYBR Green or silver nitrate to view the amplified product. This approach is usually qualitative, with analysis of the presence or absence of bands, or semi-quantitative, when densitometry of bands is used in comparison with known standards. Since it uses qualitative or semi-quantitative analysis, it is imprecise and generates false negatives with some frequency.
A variant of cPCR called "quantitative real-time PCR" (qPCR) became popular in the 2000s. It uses a quantitative approach that allows real-time monitoring of the amplification of the target PCR fragment using fluorophores that bind to double stranded DNA or linked to probes. The most commonly used method is SYBR Green: fluorophore binds to double stranded DNA molecules produced during amplification of the target fragment, leading to the emission of fluorescence during the PCR. This method has the disadvantage of not being able to directly discriminate the amplification of nonspecific DNA fragments, which is usually solved by analyzing the dissociation curve. In contrast, the TaqMan method uses a probe containing between 13 and 30 nucleotides, specifically for the target sequence and combined with a fluorophore and a fluorescence inhibitor. During polymerization of the target fragment, DNA polymerase degrades the probe and fluorescence is emitted. The use of this technique enables an increase in the specificity of this method.
Various PCR-based protocols have been developed for the detection of parasite\'s DNA and CVL diagnosis. However, the methods used may vary with respect to several parameters, such as fluorophores, probes, target regions and tissue used for detection of target DNA (Table 2), making it difficult to do a comparative analysis between the different protocols. It is known that the sensitivity and specificity of PCR for detection of
The PCR protocol sensitivity is also affected by the type of tissue used in the detection of
The selection of target region in the parasite genome is important because the variation in the number of copies, depending on the region, influences the sensitivity for detecting the parasite\'s DNA and for quantification of parasite load. The highly conserved and repetitive regions are the most commonly employed, such as the gene for subunit ribosomal RNA (rRNA) or minicircle kinetoplast DNA (kDNA) [21, 23, 127, 140, 141], that has 40-200 copies per cell, while the kDNA minicircles have about 10,000 copies distributed among 10 different classes of sequences. Using this as a target region confers high sensitivity to PCR . For quantification of the parasitic load is recommended to normalize the amount of parasite gene amplification in relation to a constitutive gene derived from the host genome in order to correct distortions caused by errors in the DNA used in the PCR reaction .
|\n \n ||\n \n ||\n \n ||\n \n ||\n \n |
|\n \n ||\n \n ||\n \n ||\n \n ||\n \n ||\n \n ||\n \n ||\n \n ||\n \n |
|Ferreira et al. 2012\n||Syber\n |
α pol DNA
|NI\n||Yes\n||Yes\n||ß - canine actin\n||(80) Infected dogs\n||Conjunctival swab, blood, bone marrow and skin\n||Comparative1,2\n||Skin > Bone marrow > Conjunctival swab > Blood\n|
|Solcà \n ||TaqMan kDNA\n||0.01 parasites/ reaction\n||Yes\n||Yes\n||18S eukaryotic rRNA\n||(51) Dogs\n||Bone marrow, conjunctival swab, lymph node, skin and spleen\n||Comparative1,2\n||Spleen > Blood > Lymph node > Skin > Bone marrow > Conjunctival swab\n|
|Belinchón-Lorenzo et al. 2013\n||TaqMan kDNA\n||0.0079 parasites/ reaction\n||Yes\n||Yes\n||18S eukaryotic rRNA\n||(28) Dogs\n||Blood, hair and lymph node\n||Comparative 2\n||Lymph node > Hair = Blood\n|
|Ferreira et al. 2013\n||Syber\n |
α pol DNA
|NI\n||Yes\n||Yes\n||ß - canine actin\n||(62) CVL positive dogs\n||Conjunctival, nasal and ear swab, blood, Bone marrow and skin\n||Comparative\n |
|Skin = Nasal swab and bone marrow > Conjunctival swab > Oral swab > Ear swab\n|
|Geisweid et al. 2013\n||Syber\n |
|NI\n||Yes\n||No\n||Canine NCX1\n||(74) CVL suspected dogs\n||Conjunctival swab, blood, bone marrow and lymph node\n||Comparative 2\n||Bone marrow > Conjunctival Swab\n|
|Reis \n ||Syber\n |
α pol DNA
|NI\n||Yes\n||No\n||G3PDH\n||(60) Seropositive dogs\n||Skin and spleen\n||Comparative\n |
|Spleen > Skin\n|
|Pennisi \n ||NI\n |
|NI\n||No\n||No\n||---\n||(6) Treated dogs\n||Blood, lymph node and skin\n||Not comparative\n||---\n|
|Francino \n ||TaqMan kDNA\n||0.001 parasites/ reaction\n||Yes\n||No\n||18S eukaryotic rRNA\n||(15) Dogs with clinical signs suggestive of CVL\n||Blood and bone marrow\n||Comparative1, 2\n||Bone marrow > Blood\n |
|Rodriguez-Cortez \n ||TaqMan kDNA\n||0.001 parasites/ reaction\n||Yes\n||Yes\n||18S eukaryotic rRNA\n||(6) Experimentally infected dogs\n||Blood, bone marrow, liver, lymph node, skin and spleen\n||Not comparative\n||---\n|
|Solano-Gallego \n ||Syber kDNA\n||7 parasites/ml\n||Yes\n||No\n||Canine GAPDH\n||(10) Symptomatic dogs naturally infected\n||Blood, bone marrow and urine\n||Comparative 2\n||Bone marrow > Blood\n |
|Manna \n ||TaqMan kDNA\n||0.001 parasites/ml\n||Yes\n||Yes\n||ß - actin\n||(18) Naturally infected treated dogs\n||Blood, lymph node and skin\n||Comparative 2\n||Lymph node > Skin > Blood\n |
|Manna \n ||TaqMan kDNA\n||NI\n||Yes\n||Yes\n||ß - actin\n||(56) Dogs\n||Blood and lymph node\n||Not comparative\n||---\n|
|Quaresma \n ||Syber kDNA\n||0.1pg DNA/ml\n||Yes\n||Yes\n||ß -canine globin\n||(35) Dogs\n||Blood and bone marrow\n||Comparative2\n||Blood = Bone marrow\n|
|Maia \n ||TaqMan\n |
|1 parasite /reaction\n||Yes\n||No\n||ß - canine actin\n||(12) Experimentally infected dogs\n||Blood, bone marrow, buffy coat, liver, lymph node, skin and spleen\n||Comparative1, 2\n||Spleen / Buffy coat / Liver / Lymph node / Bone marrow / Skin > Blood\n|
|Galletti \n ||TaqMan kDNA\n||0.03 parasite/ reaction\n||No\n||No\n||---\n||(88) Dogs\n||Conjunctival swab, Lymph node, bone marrow and blood\n||Comparative1\n||---\n|
|Lombardo \n ||TaqMan kDNA\n||NI\n||No\n||No\n||---\n||(138) Dogs\n||Blood, conjunctival and oral swabs and lymph node\n||Comparative1\n||---\n|
|Naranjo \n ||TaqMan kDNA\n||NI\n||Yes\n||No\n||18S eukaryotic rRNA\n||(22) Sick dogs\n||Main lacrimal gland, tarsal gland and nictitating membrane gland\n||Comparative1\n||---\n|
In a cytological study, Reis
Splenic collection, bone marrow and lymph node aspirates are considered invasive procedures  in addition to having an elevated cost compared to blood collection. Thus, it can be recommended to use samples obtained less invasively, such as blood and conjunctival swabs [136, 154, 155]. These samples are quick and easy to obtain, and it is low-cost compared to more invasive procedures, in addition to their higher acceptance by animal owners [132, 154, 155].
Some studies have shown that detection of parasites in the peripheral blood is less sensitive compared to other tissue samples such as spleen, bone marrow, lymph nodes and skin and tends to have variable parasitic load in accordance with the stage of infection [129, 141, 156]. However, depending on the technique and the target, blood can be used for detection of
According to Solano-Gallego
Among other less invasive sample types investigated, Solano-Gallego et al (2007) evaluated urine samples with qPCR technique, but the results described showed positivity only in dogs with severe renal injury . Naranjo et al. (2012) identified the presence of
|\n \n ||\n \n |
|\n \n ||\n \n ||\n \n ||\n \n |
|Elleviti – Torino, Italy\n||26.80*\n||---\n||63.00*\n||---\n|
|Scanelis - Toulouse, France\n||---\n||---\n||60.30*\n||---\n|
|Laboratoire d\'Anatomie Pathologique Vétérinaire du Sud-Ouest – Toulouse, France\n||---\n||---\n||---\n||127.30*\n|
|Laboratório Veterinário INNO – Braga, Portugal\n||20.60*\n||54.40*\n||---\n||---\n|
|Instituto Nacional de Investigação Agrária e Veterinária, I.P. – Lisbõa, Portugal\n||28.00*\n||41.20*\n||---\n||---\n|
|Centro de Investigación y Análisis Biológicos – Madrid, Spain\n||13.60*\n||60.30*\n||73.70*\n||---\n|
|Texas Veterinary Medical Diagnostic Laboratory – San Antonio TX, USA\n||19.20\n||---\n||---\n||---\n|
|Cornell University - Ithaca NY, USA\n||22.50\n||60.00\n||---\n||---\n|
|Hermes Pardini - Belo Horizonte MG, Brazil\n||17.20*\n||60.20*\n||---\n||---\n|
|Análisis Biológicos– Chapecó SC, Brazil\n||9.40*\n||42.15*\n||72.25*\n||--\n|
|Laborlife - Rio de Janeiro RJ, Brazil\n||30.10*\n||77.40*\n||---\n||---\n|
Despite the high sensitivity and specificity, the use of molecular methods for the CVL diagnosis presents some limitations to its use in epidemiological surveys: i) it has higher costs than other techniques (Table 3) used in the CVL diagnosis, including reagent and equipment costs; ii) it presents relative complexity in its implementation, requiring personnel with training in the execution of PCR reactions. However, this method has advantages in terms of sensitivity and specificity when compared to other diagnostic techniques, which justify its use in confirming cases screened by serology [24, 132]. Particularly due to the possibility of quantifying target DNA, qPCR may be used to monitor the parasitic load of the animal during the experimental infection, or during and after treatment in countries where it is permitted [35-37, 162]. Compared with cPCR, qPCR enables a reduction in the probability of false positives resulting from amplification artifacts and greater speed in obtaining results, once electrophoresis is no longer performed .
In summary, detailed clinical evaluation complemented with highly sensitive test allows proper identification of infected dogs in an endemic area. Evidence shows that the use of a rapid serological test associated with a molecular diagnostic test with high specificity, such as qPCR, is required for identification of all infected dogs, both asymptomatic and symptomatic. On the other hand, for sick dogs a correct diagnosis is necessary either to perform dog culling in countries where this measure is used as a control strategy of VL or to define treatment. In this case, a detailed clinical evaluation should be associated with biochemistry and hematological tests to identify signs of renal and hepatic failure, in conjunction with a serological test to confirm animal clinical condition.
Light is a major abiotic factor acts as energy source and signalling for plants growth and developments. Sun light is the prime source of energy on this planet and regulates number of essential functions in living organisms. Among them, primary producer as plants, blue green algae and photoautotrophs absorb these lights (especially 400-700 nm refereed as photosynthetically active radiation) and convert into energy source (starch, sucrose and other complex organic compound as food reserve) through the mechanism of photosynthesis . Moreover, other living organisms like heterotrophs (primary consumer and secondary consumers) fulfil their energy requirements via feeding the primary producer food reserves. Therefore, light is an essential factor for all living organism to fulfil their energy requirements. Another important function of light is to act as regulator of signalling along with growth and development functions of plant. For example, photomorphogenesis (light regulated morphogenesis), phototropism (light mediated movements), circadian rhythm (light regulated biological rhythms) and many other crucial processes are under control of light . Light characteristics such as intensity, duration, and wavelength affect the living organisms including plant in positive and negative way. Similarly, from millions of years plants are adaptive themselves to the diurnal changes of light–dark timings and many of the research concluded that change in light–dark duration affect plant growth mild to very drastic levels . However, recent advancement in human development such as highway, buildings, LEDs lightening technology, product advertisements and industries progression lead to generation of excess light in the surrounding which causes anthropogenic accelerated light pollutions. Unlike natural ecosystems, where daily activities are scheduled by natural light–dark diurnal cycles , cities are heavily lit to enable performance of a wide array of activities after dark . As artificial light at night (ALAN) becomes more reliable, efficient and affordable, living organisms become increasingly exposed to drastic and pervasive effects of “light pollution.”\n
According to Encyclopaedia Britannica, “light pollution” is “unwanted or excessive artificial light” during night hours. The introduction of ALAN into the environment majorly through electric light sources related to domestic purpose, industrial areas, transportation and street lights. The artificial night light altered the natural light cycle through its beneficial along with harmful effects on the plant ecosystem. Its beneficial impacts imply reduced the risk of night-road accident, the crime rate at night (approximately 30%), and increased working hours and scientific research areas such as speed breeding and tissue culture . Therefore, there is a need to balance the positive and negative impacts of ALAN. Biology can be a science of timing/duration, for example all organisms experience the drastic results due to change in natural light cycle variations . They are the utmost important physical factors for time. These include common daily activities (e.g. photosynthetic activity, stomatal movements, enzyme activity, flower opening, sleep movements, fragrance emission, dark recovery and repair). Indeed, the lunar cycle and the yearly/seasonal cycle dominate and regulate the lives of most organisms. Influences on biological life history have repeatedly been found to create events that occur later in life. Climatic changes can cause food and reproductive capacity to become mismatched, which in turn leads to organisms projecting highly developed phenology . Likewise, plant-environment interaction is very important to decide the present and future growth of living organisms including plants. It has been documented that ALAN affects the micro-environment attributes such as light, soil biology, humidity, biotic community and their interactions surrounding the living organisms including plant [8, 9]. Moreover, in few recent studies it was showed that ALAN not only limited to light area but it can affect the living organism beyond this and can drastically affects the ecosystem services and biodiversity . Consequently, pollination, net primary productivity, flowering, ecosystem services and nutrient recycling are the important eco-physiological functions, which are influenced by ALAN. The effect of ALAN in living organisms such as animals and humans are studied very well and several studies, and meta-analyses showed the effects on as behaviour (sleep, food, foraging, and flying), reproduction, vigilance and many other important activities [11, 12]. However, the effect of ALAN are very limited till date, only in last few years scientists are working on the ALAN and plant processes relationships and achieved some milestones. While considering the above facts, this chapter covers the present advances in ALAN research on plants specific to biorhythms and eco-physiological functions. Consequently, this analysis point out on some important strategies which are crucial for minimizing the extent of this pollution in environment.\n
Human development is continuous and complex process, also crucial for their existence for long term on this planet. This process culminates a number of novel pollutants (also known as anthropogenic pollutants) and creates a problem of other living organisms also environment sustainability. Although, this emerging pollution is global problem but the conditions might be severe when the pollution affects the endangered species in protected areas and natural hotspots . Therefore, researchers tried to quantifying the ALAN worldwide by data generated from remote sensing technology, geographical information system, hyperspectral, visual infrared imaging radiometer, day/night band, and satellites [14, 15, 16]. In the year 2001, the first globe atlas of artificial night sky brightness based on US air force satellite data, which reported that the brightness of sky is increasing continuously and in world nearly two-third of world population living in an area where ALAN is higher than threshold limits (when the artificial night light brightness is greater than 10% of natural brightness). Also, many countries such as US had this value much higher and above 99% population facing this problem . Consequently, the new world atlas on artificial night light brightness by using satellite data, day/night band, and Suomi national polar orbiting partnership satellite, which improve the resolution and accuracy reported that in new world 80% of world population facing the problem of excess night light brightness (brightness >14μcd/m2) . Therefore, ALAN is spreading swiftly and considered as global problem and now every countries trying to study the trend of light brightness in their protected area using advanced technology. For example, India analyse the trend from 1993–2013 using defense meteorological satellite programme, which help in identifying the sensitive sites such as protected areas and setting the new light policies and priorities . Although the ALAN varies in intensity, colour, timing, and wavelengths and also vary with the particular country and location. A list of ten highly polluted night light brightness cities of world and India are showed in Figure 1(a,b). In the represented figures, data on light brightness using light pollution map reveals that the situation of some cities in world such as Doha, Abu Dhabi, and Kuwait are very crucial as they have light brightness >160nW/cm2*Sr., which is 3–4 times higher than highly night light polluted brightness (50nW/cm2*Sr. considered as highly light polluted area).\n\n
The natural sky glow during night consists of comes from moon light, integrated starlight’s, zodiacal light and airglow. In the recent times due to anthropogenic activities excess light generated in the surrounding that causes disappearance of natural darkness, artificial glow in sky, and loss in visibility of the stars and Milky Way’s . There are several anthropogenic factors that are responsible for pollution in night light such as decorative lightening during various festivals, lightening in buildings, malls and in homes, traffic light (consist of four wheeler, two wheeler light), road/highway light (to reduce accidents), advertisement light (to enhance marketing of different products), ship and aeroplane light, and street lightening . These artificial lights vary in wavelength, timing (few minutes to whole night), colour (violet to red) and intensity (very low to very high). Also, the origin of this lightening varies such as LEDs light (light emitting diode), halogen light, tungsten bulb, and florescent light (tube lights). Moreover, it varies with the locations such as market area having light for 3 to 4 hours, home lights early evening hours and road light (whole night).\n
Biorhythms are known as adjustment mechanism in living systems that keep body homeostatis, adaptive processes and dynamic equilibrium. Biological rhythms are endogenous and closely linked with external environmental elements but regulated by endogenously such as genes. Phytochrome and cryptochrome pigment plays a key role in coordination and regulation of plant biorhythms [21, 22]. Phytocrome repons to red and far-red light, while crytochrome responds to ultra violet/blue light. Arabidopsis has five phytochrome
Among the all known rhythms, circadian thythms are very well known and documented by many reserchers. Circadian clocks are the biological oscillators that enable the organism to coordinate their physiology and behavior under periodic environmental fluctuations and also evolved in organisms in response to the daily rotation of the earth . Circadian clock can influence diverse plant crucial processes like leaf movement, photosynthesis, stem extension, stomatal opening and hormonal regulations . The circadian clock constitutes of three components: input, central oscillator and output pathways. Each component contains a number of genes i. e., PHYs (PHYTOCHROMEs), CRYs (CRYPTOCHROMEs) and PHOTs (PHOTOTROPINs) are the very well recognized light receptor genes of input pathways and transmit external light stimuli into the central oscillator . Phytochrome interacting factor 3 (
The circadian rhythm is closely associated to the light–dark cycle. Circadian rhythms remain consistent in response to no time cues but can be entrained by ambient conditions. But it’s been clear that different stimuli have varying effects on our circadian rhythms. Many environmental parameters provide stimulus to the clock, where the best characterized and most potent entraining stimulus is light in plants [34, 35]. Further, it is shows that shoot tip sends an unknown signal to the roots so root maintain circadian rhythm. While the lack of shoot apex signals loss of rhythmicity in the decapitated root, which can be overcome by direct exposure of root to LD cycles (light: dark) even as low intensities. It is clear that roots are entrained by light in preference to shoot apex derived signals . In the context of photoperiodism, the circadian rhythm is combined with light signaling. The photoperiod sensor permits plants to respond to the annual cycle of day length, by the production of flowers, tubers and frost tolerant buds in appropriate seasons . Accurate entrainment is important for photoperiodism, certainly general physiology shows that the crucial difference between light dominant plants (most of the flowering in long days) and dark dominant plants (most flowering in short days) is in the entrainment of their photoperiodic rhythm . In light signaling pathways both phytochrome (phy) and cryptochrome (cry) regulate clock components to attain entrainment in plants . The phytochrome responds to a red light and the cryptochrome absorbs in the UV-A/blue wavelength. Recognition of variations in day length confers plants seasonal flowering. This mechanism includes a time-keeping mechanism that integrates intimation of light environment to estimate the duration of day or night. Time-keeping activity is the outcome of the circadian clock. In Arabidopsis, an increase in flowering under long days (LD) happens through transcriptional induction of florigen gene FLOWERING LOCUS T (FT) specifically under LD conditions. The FT promoter binds with CONSTANS (CO) transcription factor which directly confer its LD-specific induction . CONSTANS characterizes as a photoperiodic timer gene with its transcript level being managed by the circadian clock. CO proteins accumulate in response to exposure to light .\n
As sessile plant receive light as signal and resource. Diurnal cycle of dark and light period is vital for regulating numerous processes in plants such as daily events of enzymes activity, gas exchange, photoperiodic movements, metabolism, stomatal movements, flowering opening, dark repair recovery and photosynthesis. The impact of artificial lighting on the body clock and its circadian rhythms is an important research topic. Night shifts introduction has changed the natural 24-hour cycle. There are multiple different categories of artificial lighting that are being utilized. It is various forms, from brief pulse to long lasting night glow, from narrow emission spectra to broad emission spectra, from low emission intensity to high intensity emission, and from local emission focus to glowing of sky. Effect of ALAN on some plant circadian process are disused thoroughly and represented in Figure 2.\n\n
It is very well documented that enzymes are essential for optimum growth and development of plant. Also, they help in regulating all the physiological, biochemical processes in plants. Important physiological and biochemical processes of plant include such as seed germination, photosynthesis, respiration, and translocation. In this regards an experiment conducted in rice using the different light intensity (from low to high) during night hours and observed that ALAN drastically reduce the germination capacity by reducing the activity of α-amylase enzyme . Moreover, a recent study conducted perennial ryegrass using the different duration of light as light/dark hr. (24/0, 22/2, 20/4, 18/6, 16/8, and 14/10) and they had found that seed germination percentage (67 to 33%), soluble carbohydrates (27.48 to 9.16 mg/g fresh weight) and soluble protein (13.85 to 10.59 mg/g fresh weight) are lowest in 24/0 conditions as compared to 16/0 light/dark conditions, which showed the drastic effect on the future growth of plant . In this array, a study conducted in yellow poplar using different light intensity from low 1 to high 50 μmol m−2 s−1 showed that in ALAN leading to reduction in starch turnover (74.7 to 11.4%) via affecting the starch synthesis activity and lower rate of respiration [44, 45]. Therefore, from these studies, it can be concluded that ALAN affect the metabolism of plant at early and late growth stages.\n
The stomata are very important for gas exchange and water transpiration in plants. It is also well documented that stomatal movement is circadian rhythm which is also influenced by the intensity and quality of light. In this regard, an experiment conducted using different photon flux density (1, 3, and 50 μmol m−2 s−1) and different light sources low pressure sodium lamp, high pressure sodium lamp and LEDs and they found shorter, narrower stomatal aperture, change in osmotic pressure and reduced stomatal size with increased stomatal density and the higher intensity more drastically affect the stomatal attributes in comparison with lower intensity of ALAN. Further, it is also found that the stomatal conductance is reduced under ALAN condition and affects the biomass accumulation and gas exchange [44, 45]. ALAN reduces the both above and below ground fresh and dry weight. Such as in the presence of 50 μmol m−2 s−1 in ryegrass during night time reduces the below ground fresh and dry weight by 48 and 46% respectively. Similarly, the reduction in above ground fresh and dry weight are 27 and 46% respectively. Therefore, it is expected that in future the result of ALAN might be more drastic and influence stomatal behaviours in plants.\n
Photosynthesis is the most crucial physiological process in autotrophs and essential for sustaining life on this planet. Earlier, it is documented that the sunlight is the prime source of energy for photosynthesis processes but in recent years many studies conducted using ALAN and found that it hampers the photosynthetic processes. In this aspect a recent experiment conducted using the street light having ALAN intensity near to 340-360lux and exhibited that plant under street light have lower photosynthetic quantum yield (Fv/Fm), non-photochemical quenching (NPQ) values, which ultimately affects the photosynthetic rate drastically . Similarly, a study conducted in yellow poplar using different light intensity and quality during night hours reveals that total chlorophyll content reduced up to 35%, water use efficiency 23%, and photosynthetic rate 42 to 45% . Another study reported in yellow poplar showed that the ALAN affect the ultrastructure of chloroplast by multiplying number of thylakoid membrane stack, reducing thylakoid stacking, and increasing number and size of plasoglobuli . These modifications in chloroplast ultrastructure lead to early senescence, chloric and abnormal leaves. Consequently, ALAN in ryegrass influences the Chl a, chl b and total chlorophyll content, Fv/Fm ration, and electron transport chain. Further, they also ended up that the ALAN act as stressor and influence the photosynthetic efficiency of plants . Therefore, it can be concluded that ALAN act as a stressor for photosynthetic phenomena and expected that in future it may be a global problem of road side trees.\n
The impact of relative length of flowering during day and night is refereed as photoperiodism. On the basis of day and night length plants are categorised into long day plant (required longer day length than critical period), short day plant (required shorter than crucial photoperiod) and day neutral plant (do not affect by day length). Therefore, flowering mechanism is very sensitive to critical day length. In the recent years it is found that ALAN severely affects the flowering mechanism in plants. For example, under ALAN and average spring temperature conditions at timing of budburst in deciduous tree species recorded and observed the budburst occurrence about 7.5 days earlier as compared to the normal UK conditions . A long term experiment conducted to observe the impact of ALAN on wild species in natural and semi-natural grassland and authors found that the lightening affects the trajectory of vegetation cover, leading to change in plant biomass and the composition of dominant wild species. Further, they also suggested that the ALAN significantly alters the flowering phenology by shortening or lengthening of flowering period varies from 4 day earlier to 12 day late compared to control conditions . Nevertheless, authors observed the non-significant relationship of ALAN with vegetation composition and flower density in grassland vegetation species. Further, they observed early flowering (about 4 days) in
As earlier mentioned ALAN is a global problem and it act as a stressor for living organisms including plant. Also, some recent studies exhibited that the impact of ALAN can be amplified with unfavourable conditions as pollution. Moreover, the living organisms or plants connected with environmental phenomenon and adaptive themselves. Many plant processes are associated to environment such as transpiration which depends upon the atmosphere humidity. Some of eco-physiological processes influenced by ALAN condition are followed and represented in Figure 2.\n
It is very well documented that the growth and development of plant is highly influenced by the environmental conditions. Also, a positive plant-environment interaction is necessary to grow plant under different environmental circumstances. The night hour lightening has great impact on the plant phenology, physiology, growth and development, reproduction and behaviours . The light pollution is an emerging global phenomenon and accelerated by rapid urbanization, which affects both plant and animal fitness in both developed and developing countries, however the impact are very serious in urban areas. Contrary, it is observed that delayed autumn phenophases in the crown part of two tree species (
Globally, pollinators are indispensable source of pollination in both agriculturally important crops and wild plants . Anthropogenic activities such as intensive agriculture, use of extensive pesticides, habitat change, invasive alien species and climate change has substantially reduced population of pollinators . Moreover, ALAN has emerged a new threat to plant reproductive success by disturbing plant-pollinator ecosystem balance. For example, authors witnessed the reduction of nocturnal pollinators visit by 62% in ALAN areas compared to the dark areas and yield was reduced by 13% . They also observed visit of diurnal pollinators at night with nocturnal pollinators under ALAN. The pollinator-population dynamics and plant-pollinator communities are being adversely affected by artificial night lightening. Furthermore, ecological functionality and stability has been challenged by artificial night lightening throughout the world. Also the pollution effect of ALAN by nocturnal moths (Lepidoptera) and concluded that pollination is an ecosystem driven process that can be disturbed by increase of light pollution . Similarly, recent study observed the negative relationship between direct ALAN and reproductive out in
As sessile in nature plant faces a number of stressor throughout their life cycle such as abiotic stress (drought, heat, flood, cold, salinity, elevated CO2, and heavy metals), pollutions (air pollution, soil pollution), biotic stresses (insect, and pathogen), and most of times these stress are in combined from and cause for detrimental loss in their optimum growth and developments. All of these are act as stressor for plant and affect plant by changing the physiological, biochemical and molecular processes . Likewise, this light also an important environmental factor and low and high light both act as stressor [42, 61]. Recent studies shows that ALAN also act as stressor and found out that it increases the lipid peroxidation, and reduces total antioxidant capacity in autotrophic red sea corals . Further, an experiment in perennial ryegrass showed that ALAN increases the malondialdehyde (MDA) content and reduce leaf heat stability . Similarly, it is observed that yellow poplar plant accumulate hydrogen peroxide, superoxide radicles and reduced abscisic content under ALAN conditions [44, 45]. Therefore, from these studies it may be concluded that ALAN act as a stressor and responsible for oxidative stress.\n
Abiotic and biotic component of ecosystem coordinate to each other which provide important ecosystem services to humans. Also, the stability of ecosystems is key for sustaining of life on this planet. However, in the recent past due to uncontrolled anthropogenic activities ecosystem and biodiversity services of ecosystems are losing continuously. Among the anthropogenic activities, ALAN is one of the swiftly expanding activity, which is now become a global problem for ecosystem and biodiversity services [10, 63]. It is suggested that ALAN affect the organisms flux across the ecosystems and key driver of ecosystem community structure and can modify the ecosystem functioning beyond the affected area . Moreover, the effects of ALAN are not limited to plants but also it affects the aquatic, forest, desert, terrestrial, mountain and agriculture ecosystem from lower to higher levels [10, 59](. Further it has been found the ALAN had drastic impact of ecosystem services such as foraging, vision, reproduction, signalling and behaviour. Therefore, it can be concluded that ALAN had serious impact on ecosystem services of stable ecosystems.\n
Street lights should be replaced with energy-efficient LEDs and proper shielding of light in streets and by managing their angels. Street lights should be installed where needed Indian standards  has classified the roads according to the traffic density. By considering the road category according to traffic density engineers can design the street light plan and provide the installation specifications. To a report from , the Bruhat Bengaluru Mahanagara Palike (BBMP) projected a plan to replace the existing 4.8 lakh city street lights with high energy-efficient LED lights. A number studies conducted to showing their impact on plants ecosystems and hot spots and most of cases it affect in negative way. Although, some of researcher are concerned and documented about the impact of ALAN but there is no progress in changing the street lights. However, in recent time some countries follow the new light policies, where they replaced the old lights with more efficient lights and change the angle and height of light source to reduce the tree passes.\n
This can be minimize using smart building architecture to decrease the use of excessive outdoor and indoor lights. The utilisation of LEDs, compact fluorescents (CFS) and warm coloured bulbs should be used to minimise energy use and by this way somehow we can protect our environment. The dimmers, motion-sensors and timers contribute a lot to energy saving. Turning-off the unnecessary indoor along with outdoor lights in houses and offices. Avoid Blue light during night time. International Dark Sky-Association (IDA) recommends use only warm lights for outdoor that includes low-pressure sodium (LPS), high-pressured Sodium (HPS) and low-colour LEDs. By using warm or filtered LEDs (CCT < 3,000 K; S/P ratio < 1.2) can reduce the blue light emission .\n
Artificial light pollution is now emerging pollution and governments has also considered it and have provided certain guidelines to decrease the energy wastage, damage to agro-ecological and wildlife ecosystem. During the 13th meeting of the Conference of the Parties, Gandhinagar, India Agenda No. 26.4 “the guidelines for light pollution for wildlife including marine turtle, seabirds and migratory shorebirds” under the Convention on the migratory species (prepared by the Government of Australia) . By seeing the threat of sky glow and emerging challenges of increasing artificial light pollution to the marine species government has taken a proactive approach to develop artificial light in night pollution guidelines. These kinds of initiatives need to be taken by every country to combat artificial night light pollution. Artificial light at night became an unavoidable technology from the societies however this indispensable tool has harmful side effect in term of light pollution . One can understand that artificial light at night is necessary to highlight the scenic beauties of the cities and to provide safety on roads however where artificial light at night is easily available at very low cost there it is over utilized which leads to discomfort in society with a view to disturbing the natural habitats, underline the beauty of skyline etc. Therefore now we can understand that ALAN has two phases of its representation. The detrimental effect can be seen through alteration in the sky glow which is an important constituent of biosphere . According to one study, around one fifth of the global population affected by artificial sky brightness . Challenge that we have is how we can use the ALAN in such a way that maximize the social benefits and reduce the impact of its pollution.\n
In this regards several articles are available at public domain [71, 72, 73, 74, 75, 76, 77, 78, 79]. They have summarized the solution like protecting species rich areas in their natural habitat, policy making on threshold and upper limit of light emission, environmental specific custom based light brightness which is adapted to vicinity of that area, time control of light emission, diurnal adapted colour spectrum and reducing the trespass of lightening. Apart from this, it is suggested that the five ways to minimize the harmful effect of ALAN
Reduce the height of light source to reduce tress passes
Change the angle and position of light source
Light source could be away from trees
Growing light insensitive tress and crops o road side areas such as day neutral species
Avoid short day plants near to highways
Promote self-pollinated species as compared to cross pollinated
Avoid cross-pollinated species especially which nocturnal pollinated
This approach can be milestone to reduce the impact of ALAN on plants.\n
Human population is continuously expanding and degradation of natural resources is also increasing with human development activities. ALAN has important role during the human development process as it increases the working hours, work efficiency, reduce crime and accidents but due to excess use and accumulation in environment cause a problem of artificial night light pollution. ALAN pollution is anthropogenic and increasing swiftly and has global impact. In the presented chapter we tried to explore the impact of ALAN on plant biological rhythms and ecophysiological processes. Therefore, this study comprises of the recent status of ALAN in world and their sources. In this we concluded that many countries are facing the problem of ALAN and continue expanding their area. Further, we discussed about the plant biorhythms and their regulation by diurnal cycles and concluded that the plant biorhythms are highly sensitive too change in light/dark periods. Thereafter, we provide the some details regarding the plant circadian rhythm, which are affected by ALAN and concluded that ALAN had negatively associated with them. Later on, we detail the some ecophysiological functions under ALAN and concluded that ALAN had drastic effects on plant processes and in future it can be a global problem. In the last we discussed the some strategies and approaches to minimize the effect of ALAN. Therefore, in this chapter we tried to comprise all the recent information, which help scientist to explore more about in this area of research.\n
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