Canine heartworm in the domestic and wild canids of southeastern Nebraska

The prevalence of canine heartworm (Dirofilaria immitis) was examined in the domestic dog, coyote (Canis latrans), and red fox (Vulpes fulva) populations of southeastern Nebraska. Microfilariae were detected in 21.4% (22 of 103) of the domestic dogs. The average age of infection for dogs was 5.8 yr. Nine of the 22 infected dogs also were positive for Dipetalonema reconditum. Thirty-nine of 443 (8.9%) coyotes were found to have adult heartworms. The average number of male and female worms per heart was 3.7 and 3.9, respectively. The mean age of infected coyotes was 3.6 yr. Red foxes had an infection rate of 4.8% (1 of 21). This fox heart had only 1 immature female worm. A mail survey of 6 veterinary clinics was also conducted. Veterinarians reported infection rates of 0% to 18.8% in domestic dogs for their localities.     

Effect of host sex and litter on the population dynamics of Echinococcus granulosus in dogs.

Twenty-one Beagle dogs consisting of 10 males and 11 females and belonging to 3 litters were infected with 60,000 E. granulosus protoscolices each. They were killed on day 40, the parasites from their intestines recovered, and the number of worms, average number of proglottides per worm, average length per worm, percentage of worms with a uterine cavity, and percentage of egg-bearing worms were determined for each dog and analyzed per sex and litter. On average, the dogs had 1,253 +/- 339 worms (means +/- standard error) with 2.42 +/- 0.1 proglottides, were 1.59 +/- 0.07 mm long, and 25.6 +/- 4.8% of the worms presented a uterine cavity and 1.2 +/- 0.6% bore eggs. The number of worms exhibited a bimodal distribution with 19 dogs having less than or equal to 2,565 worms and 2 greater than or equal to 5,520 worms. Average number of proglottides also showed a bimodal distribution with 7 dogs having less than or equal to 2.1 proglottides per worm and 14 dogs having greater than or equal to 2.4 proglottides per worm. The parasites were significantly more numerous in females than in the males (1,964 +/- 573 vs. 681 +/- 202), had more proglottides (2.67 +/- 0.08 vs. 2.15 +/- 0.16), and were longer (1.72 +/- 0.07 vs. 1.44 +/- 0.11 mm). The percentages of parasites with a uterine cavity (27.8 +/- 5.9 vs. 23.2 +/- 8.1) or bearing eggs (1.0 +/- 0.5 vs. 1.5 +/- 1.8) were comparable in females and males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations

A sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Echinococcus multilocularis coproantigens (EM-ELISA) was developed with polyclonal rabbit (solid phase) and chicken egg (catching) antibodies that were directed against E. multilocularis coproantigens and somatic worm antigens, respectively. In experimentally infected dogs and cats, coproantigens were first detectable 6-17 days postinfection (PI) in samples of 8 dogs (worm burdens at necropsy: 6,330-43,200) and from 11 days PI onward in samples of 5 cats infected with 20-6,833 worms. After anthelmintic treatment of 4 dogs and 5 cats at day 20 PI, coproantigen excretion disappeared within 3-5 days. The sensitivity of the ELISA was 83.6% in 55 foxes infected with 4-60,000 E. multilocularis, but reached 93.3% in the 45 foxes harboring more than 20 worms. The EM-ELISA was used in surveys of "normal" dog and cat populations in Switzerland. Among 660 dogs and 263 cats, 5 dogs and 2 cats exhibited a positive reaction. In 2 of these dogs (0.30%) and 1 cat (0.38%), intestinal E. multilocularis infections were confirmed by necropsy, polymerase chain reaction PCR, or both. The specificites of the ELISA in these groups were found to be 99.5% and 99.6%, respectively, if positive ELISA results that could not be confirmed by other methods were classified as "false positive" reactions.     

The use of excretory and secretory antigens of the scolex of Taenia ovis for the serodiagnosis of infection in dogs

The excretory and secretory antigens from the evaginated scoleces of Taenia ovis were collected for 3 days in vitro, and used in an ELISA test to detect antibodies to T. ovis in the serum of dogs. When tested on sequentially collected sera, diagnostic ELISA values could be detected in many dogs 4 wk after infection, and remained for an average of a further 4 wk after worms were removed from dogs with an anthelmintic. Using an ELISA discriminant value that eliminated all false positives from 70 uninfected laboratory dog sera and from 57 uninfected farm dog sera, 54/62 true positives were found in sera from dogs infected with various numbers of T. ovis for various intervals. Sera from dogs infected with T. hydatigena gave 11/15 false positive reactions, whereas sera from 15 dogs infected with Echinococcus granulosus or 7 dogs infected with T. pisiformis were all negative. For T. ovis the test had a high repeatability, was not greatly influenced by the number of worms carried by the dog and higher titres were correlated with long-standing infections. Approximately 1,000 scoleces could be recovered from each experimentally infected sheep. Using the ELISA test with undiluted antigen and serum diluted 1:40, approximately 10 sera could be tested in duplicate with the excretions and secretions from each T. ovis scolex.     

First record of Anguillicoloides crassus (Nematoda) in American eels (Anguilla rostrata) in Canadian estuaries, Cape Breton, Nova Scotia

In the summer of 2007, American eels, Anguilla rostrata, from 2 localities on Cape Breton Island, were found to be infected with the swim bladder nematode Anguillicoloides crassus. This is the first documented report of this highly invasive parasite in Canadian waters. More than half of the yellow eels in Mira River (6 of 10), and 1 eel (of 5) from Sydney Harbour were infected. Parasite intensity ranged from 1 to 11 worms per eel. The occurrence of A. crassus at these 2 localities suggests the need for a more extensive survey on the distribution of this exotic parasite in eel populations throughout Cape Breton Island.     

Diagnosis of canine echinococcosis: comparison of coproantigen detection with necropsy in stray dogs and red foxes from northern Jordan

The sandwich enzyme linked immunosorbent assay (ELISA) was used as a diagnostic test for Echinococcus granulosus infection by detecting coproantigens in 94 stray dogs Canis familiaris and eight red foxes (Vulpes vulpes) from northern Jordan. The results were analyzed in relation to actual helminth infection as revealed by necropsy. The infection rate of dogs with E. granulosus was 13.8% with a worm load ranging between 3-> 10,000 per infected dog. In contrast, eight of 13 E. granulosus infected dogs were coproantigen positive (overall sensitivity 61.5%). The sensitivity increased to 87.5% and 100% in dogs harboring > 20 and > 100 worms/dog, respectively. The specificity of coproantigen-ELISA was 91%. The greatest cross-reactivity was found in dogs infected with Dipylidium caninum. The positive and negative predictive values for the coproantigen-ELISA test were 50% and 94.2%, respectively. Thus, a coproantigen negative dog is most probably truly negative for E. granulosus. In contrast, a coproantigen positive dog may not be truly positive for E. granulosus, except if it has a high worm burden of > 100 worms/animal.     

Prevalence of canine dirofilariasis in Taiwan

Between 1993 and 1997, 837 stray dogs from North Taiwan were necropsied and examined for dog heartworm infections. A thick smear from 20 ml of peripheral blood from each dog was also prepared and examined for microfilariae (mf). The overall prevalence of adult worms in the dog population was 57%. The prevalence of mf in 1228 house dogs from different parts of Taiwan was also determined from 20 ml of peripheral blood in the same way. The overall prevalence of mf was 25%, with a value of 30% in the main island of Taiwan, this being 15 times higher than that in the offshore islands (2%). In Taiwan, the prevalence ranged from 4% in Hualien County, East Taiwan, to 41% in Nantou County, Central Taiwan. The mf prevalences on offshore islands were 1% on Liuchiu and 2% on Lanyu. The mf density per 20 ml blood in 82 house dogs was found to be 23 mf per dog, with a range of 3-97 mf per dog. A total of 477 stray dogs were found to be infected with adult worms of Dirofilaria immitis. The mean number of 7 worms per dog was obtained, with a range of 1-55 worms per dog. These results indicate that the prevalence of canine dirofilariasis has increased in Taiwan over the past 10 years. Moreover, the prevalence may be related to the wind speed, temperature, relative humidity, and altitude in the different areas surveyed.     

Detection of Echinococcus granulosus coproantigens in Australian canids with natural or experimental infection

Coproparasitological and purging methods for diagnosing canids infected with the intestinal helminth Echinococcus granulosus, an important zoonotic parasite, are unreliable. Detection of coproantigens in feces of infected dogs by enzyme-linked immunosorbent assay (ELISA) is suitable for detecting patent and prepatent infections with a high degree of sensitivity and specificity. In the present study, natural and experimental infections in domestic and wild Australian canids were investigated using a coproantigen capture ELISA. Experimental infection of dogs with E. granulosus was detected at between 14 and 22 days postinfection (PI), and optical density (OD) values remained high until termination of experiments 35 days PI. After chemotherapy, coproantigen levels in infected dogs dropped rapidly, becoming negative 2-4 days after treatment. In experimentally infected red foxes (Vulpes vulpes), the coproantigen excretion profile was different, with ELISA OD levels peaking 15-17 days PI, then falling to low or undetectable levels by 30 days PI. Coproantigens were detected in the feces of naturally infected Australian wild dogs (dingoes, dingo/domestic dog hybrids) with infection levels ranging between 2 worms and 42,600. Preliminary data on the stability of coproantigen in dog feces exposed to environmental conditions indicated that there was no change in antigenicity over 6 days. The results suggest the coproantigen ELISA could be successfully used to monitor E. granulosus prevalence rates in Australian domestic dogs, foxes, and wild dogs.     

Antibody responses against natural Taenia hydatigena infection in dogs in Kenya

Antibody responses (IgG) against Taenia hydatigena infection in dogs in Kenya were analysed in ELISA using excretory/secretory products of T. hydatigena scoleces derived from goat cysticercus cysts. Helminth infections of individual dogs were confirmed at autopsy. T. hydatigena worms were found in 89.5% of 143 dogs, and positive anti-T. hydatigena antibody levels were detected in 58.7% of infected dogs. Positive antiscolex antibody levels were detected in 40.0% of Turkana dogs uninfected with T. hydatigena, suggesting previous infection. Antibody was not detected in 34.4% of infected dogs. There was no relationship between individual T. hydatigena worm burdens and absorbance values for sera in ELISA. It was not possible to distinguish between sera from T. hydatigena-infected and uninfected dogs.     

Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats

A total of 15 red foxes, 15 raccoon dogs, 15 domestic dogs and 15 domestic cats were each infected with 20,000 protoscolices of Echinococcus multilocularis. At 35, 63, and 90 days post inoculation (dpi), five animals from each group were necropsied and the worm burdens determined. The highest worm burdens in foxes (mean of 16,792) and raccoon dogs (mean of 7930) were found at 35 dpi. These declined to a mean of just 331 worms in foxes and 3213 worms in raccoon dogs by day 63 with a further decline to 134 worms in foxes and 67 worms in raccoon dogs by day 90. In dogs, there was no significant difference between worm burdens recovered at days 35 (mean of 2466) and day 90 (mean of 1563), although reduced numbers were recovered on day 63 (mean of 899). In cats, worms were found in four animals 35 dpi (mean of 642), in three at 63 dpi (mean of 28) and in two at 90 dpi (mean of 57). Faecal egg counts were determined at 3 day intervals from 25 dpi. A mathematical model of egg excretion dynamics suggested that the mean biotic potential per infected animal was high in foxes (346,473 eggs); raccoon dogs (335,361 eggs) and dogs (279,910 eggs) but very low for cats (573 eggs). It also indicated that approximately 114, 42 and 27 eggs per worm were excreted in the faeces of dogs, raccoon dogs and foxes, respectively. The fecundity of worms in cats was low with an average of less than one egg per worm. The peak levels of coproantigen were detected earlier in foxes and raccoon dogs than in dogs. Eggs recovered from foxes, raccoon dogs and dogs resulted in massive infections in experimental mice. However, metacestodes did not develop from eggs originating from infected cats. It is concluded that foxes, raccoon dogs and dogs are good hosts of E. multilocularis. In contrast, the low worm establishment, the very few excreted eggs and the lack of infectivity of eggs strongly indicate that cats play an insignificant role in parasite transmission.     

Efficacy of ivermectin in hookworms as examined in Ancylostoma caninum infections

Single oral doses of ivermectin were given to dogs with moderate or heavy infections of Ancylostoma caninum (egg counts ranging from 7,100 to 41,700 eggs/g feces) at 100, 50, 30, or 10 micrograms/kg body weight. Each of these dosages was effective in clearing the infection completely, so that numerous worms were passed in the feces on days 1-3, but no worm was recovered from the intestinal tract at necropsy on day 4 after treatment. In contrast, an average of 178 worms per dog was recovered at necropsy from the vehicle-treated control and the untreated animals. Albendazole, a known anti-hookworm agent, even in a dose of 400 mg, eliminated only 21-65% of the worms harbored by the infected animals. No untoward reaction to ivermectin or significant pathological change was noted in the experimental animals. In vitro experiments demonstrated that ivermectin: (1) was highly detrimental to actively motile adult worms in concentrations greater than 5.60 micrograms/ml; (2) was detrimental to eggs inside the uterine tissue of female worms in dosages at or greater than 10 micrograms/kg body weight; and (3) killed infected larvae in concentrations as low as 0.0025 micrograms/ml.     

Experimental infection of Dirofilaria immitis in raccoon dogs

Canine heartworm (Dirofilaria immitis) is a nematode that naturally parasitizes in the pulmonary arteries and the right ventricle of domestic dogs (Canis familiaris) as final hosts. Japanese raccoon dogs (Nyctereutes procyonoides viverrinus) also are known to be susceptible to infection by the parasite. However, prevalence of this infection among free-ranging raccoon dogs is low and so is the worm burden. To examine the susceptibility of the raccoon dog to D. immitis infection, 3 raccoon dogs and 2 beagles were inoculated 4 times with 25 third-stage larvae (L3s) of D. immitis at 3-wk intervals. Worms were recovered from 2 raccoon dogs and both domestic dogs. The average percentage of recovery (2.3%) of the raccoon dogs was almost 10 times lower (24.5%) than that of the domestic dogs, but there was no significant difference in the body length of worms recovered from 2 types of hosts. To examine microfilaremia, 2 raccoon dogs were infected with 100 L3s. Microfilaremia was observed for 180 days postinoculation (PI) but disappeared at about 300 days PI. The raccoon dog was mildly susceptible to infection with D. immitis, but surviving worms developed and matured normally.     

Correlation between discharged worms and fecal egg counts in human clonorchiasis

Background

Stool examination by counting eggs per gram of feces (EPGs) is the best method to estimate worm burden of Clonorchis sinensis in infected humans. The present study investigated a correlation between EPGs and worm burden in human clonorchiasis.

Methods and Findings

A total of 60 residents, 50 egg-positive and 10 egg-negative, in Sancheong-gun, Korea, participated in this worm collection trial in 2006–2009. They were diagnosed by egg positivity in feces using the Kato-Katz method. After administration of praziquantel, they were purged with cathartics on the next day, and then discharged adult worms were collected from their feces. Their EPGs ranged from 0 to 65,544. Adult worms of C. sinensis were collected from 17 egg-positive cases, and the number of worms ranged from 1 to 114 in each individual. A positive correlation between EPGs and numbers of worms was demonstrated (r = 0.681, P<0.001). Worm recovery rates were 9.7% in cases of EPGs 1–1,000 and 73.7% in those of EPGs over 1,000. No worms were detected from egg-negative subjects. Maximum egg count per worm per day was roughly estimated 3,770 in a subject with EPGs 2,664 and 106 collected worms.

Conclusions

The numbers of the worms are significantly correlated with the egg counts in human clonorchiasis. It is estimated that at least 110 worms are infected in a human body with EPGs around 3,000, and egg productivity of a worm per day is around 4,000.     

Canine heartworms in coyotes in Illinois

Canine heartworm (Dirofilaria immitis) disease affects wild canids and may be a factor impacting the health and population dynamics of coyotes (Canis latrans). Coyotes may serve also as a potential reservoir for transmission of these parasites to domestic dogs. We investigated 920 coyotes harvested by hunters and trappers throughout Illinois (USA) from 1995-1997. The objectives of the study were to: 1) survey the regional prevalence and intensity of heartworms in coyotes in Illinois, 2) determine whether heartworm intensity correlates with physical condition, particularly body weight and winter fat levels, and 3) evaluate the relationship between heartworm infections and the reproductive success of females. Prevalence of heartworms statewide was 16.0%. Prevalence was significantly higher in males (17.7%) than in females (14.1%; P = 0.04) and was higher in the older age-classes (P < 0.0001). The regional prevalence of heartworms increased from northern to southern Illinois. Intensity ranged from 1 to 111 with a mean of 8.7 (SD = 13.2) worms. Intensities did not differ significantly between sexes (P = 0.53) or among age-classes (P = 0.84). Most infected coyotes had low intensity infections, 78.2% carried < 12 heartworms, 11.6% had 12-24 worms, and 10.2% were infected with > 24 worms. Body weights were not correlated with the presence of heartworms, nor were levels of kidney fat and marrow fat. However, reproductive success was lower in infected females. The percent of yearling females that bred was lower among infected females, as was the number of offspring produced by adults > or = 3.5 yr old. Our study demonstrates that heavy infections adversely affect fur quality and reduce fecundity of some females, but these effects are small and few coyotes (4.1%) had enough worms to trigger them. Coyote populations have increased in Illinois during the past 20 yr, but prevalence and intensity of heartworm disease appears to have changed little in that period. We conclude that heartworm disease is only a minor factor influencing coyote population dynamics in Illinois.     

Concurrent infections of Echinostoma caproni and Echinostoma trivolvis in ICR mice.

ICR female mice, 6- to 8-weeks old, were exposed concurrently to 25 metacercarial cysts of Echinostoma caproni and 25 metacercarial cysts of Echinostoma trivolvis and necropsied 10 and 14 days post-infection. Controls consisted of mice exposed singly to either 25 or 50 E. caproni or E. trivolvis cysts. All 23 mice exposed to E. caproni cysts were infected with a total of 331 worms (37.8%), whereas only 11 (37.9%) of 29 mice exposed to E. trivolvis cysts were infected with a total of 77 (6.4%) worms. In the concurrent infections, 13 (59.1%) of 22 mice were infected with both species and the percentage of worm recovery was 72.6% for E. caproni and 14.2% for E. trivolvis. There was no difference in worm distribution of either species in single vs concurrent infections. In concurrent infections at 14 days PI, there was a significant decrease in the body area of worms of both species, when compared to single worm species.     

Prevalence of Enterobius vermicularis infection and preventive effects of mass treatment among children in rural and urban areas, and children in orphanages]

An epidemiological study and mass treatments of Enterobius vermicularis infection among children near Wonju area of Kangwon province were carried out. The children were divided into 4 groups according to their residing localities; children in the mountainous area, rural area, urban area and in orphanage. They were examined by adhesive cellotape anal swab technique, and egg positive rates were obtained. The rates of egg reduction and re-infection rates after repeated mass treatments were also observed. The results obtained were as follows: 1. The overall egg positive rate of E. vermicularis in the first screening was 19.9% (251 out of 1,262 examinees; 19.7% in males and 20.1% in females). The positive rates were 13.0% in the mountainous area, 11.9% in the rural area, 15.1% in the urban (medium-sized) area and 61.9% in orphanages. 2. The highest positive rates were observed in the kindergarten children, and 1st and 2nd grade children of primary schools (26.2-32.2%), and the lowest rate (13.6%) in 6-year grade children of primary schools. 3. Cumulative detection rates from 3 repeated anal swabs at 4-5 days interval were higher (70.8%) than those from single anal swabs (50.0-59.2%). 4. Out of the examinees who showed the highest cumulative positive rate (70.8%), about 39.2% were consecutively positive in 3 anal swabs. Among different groups of children, the higher the total egg detection rates (87.5%), the higher the consecutive positive rates (71.9%). 5. A total of 2,609 (male: female = 1:12.4) worms were collected from 17 egg-positive cases treated with anthelmintics. The mean number of worms per child was 153 (range: 4-824).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence of Hydatid Cysts in Livestock Animals in Xinjiang,China

Hydatid worms, hosted by humans and animals, impose serious human health risk and cause significant livestock production loss. To better understand the disease infection status in Xinjiang, China, we investigated the disease epidemics in 4 livestock animals, i.e., cattle, sheep (both sheep and goat), camels, and horses, slaughtered at the abattoirs in Urumqi, Yining, Tacheng, and Altay areas. The results showed that the animals were infected at different rates, in the order of sheep (9.8%), cattle (8.4%), camels (6.8%), and horses (4.3%). The infection rates were found to be different between the abattoirs in various regions even for the same animals. For sheep, the rates increased significantly as the animals grew older. It was 1.9% before 1 year of age and increased to 8.2% in the age of 1-2 years, and further increased to 12.3% when the animals were 3-4 years old, and reached 17.2% when they were 5-6 year old. Sheep older than 6 years had an infection rate of 19.5%. This study demonstrates that the 4 livestock animals in the pastoral areas in Xinjiang were infected by the parasites to various extend. This study is the first systematic investigation of the hydatid worms in various livestock animals in Xinjiang, China, which provides epidemiological information about the infection of hydatid worms in livestock, and is valuable in developing strategies for prevention and control of the hydatid disease.     

Coproantigen detection in dogs experimentally and naturally infected with Echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay

A sandwich ELISA for the detection of Echinococcus granulosus coproantigen in formalin and heat-treated faecal supernatants of dogs was developed. The assay used affinity-purified polyclonal antibodies obtained from rabbits hyperimmunised with E. granulosus excretory/secretory antigens and biotiaylated monoclonal antibody EmA9 produced against adult E. multilocularis somatic extract. The test was sensitive to 7 ng and 2.3 ng of E. granulosus protein and carbohydrate/ml of faecal supernatant, respectively. Thirteen helminth-free dogs were infected with different amounts of E. granulosus protoscoleces and the presence of coproantigen was monitored during the prepatent period until day 35 post-infection, when they were necropsied. Faecal antigen levels started to rise above the normal range between days 10 and 20 post-infection, and typically peaked at the end of the experiment. All the dogs, bearing from 3 to 67 700 worms, showed positive values in the ELISA during the prepatent period. One dog experimentally infected with Taenia hydatigena metacestode and harbouring three worms, tested positive only after the prepatent period at day 52. The test was applied to 98 stray dogs. The ELISA detected all of four dogs naturally infected with E. granulosus, two dogs with patent infections of T. hydatigena and two dogs with no cestode infections, sbowing a sensitivity of 100% and a specificity of 96%.     

New records of nematomorph parasites (Nematomorpha: Gordiida) of ground beetles (Coleoptera: Carabidae) and camel crickets (Orthoptera: Rhaphidophoridae) in Washington State

From 1998 to 2003, beetles and crickets infected with hairworms were collected from 4 localities within the Hanford Nuclear Site and the Hanford Reach National Monument, located in a shrub-steppe region of Washington State along the Columbia River. Infected hosts comprised 6 species of carabid beetles within 5 genera and 2 camel crickets within 1 genus; all are newly documented insect-nematomorph associations. A large proportion of the infected hosts (48%) were collected from a single site during a single collecting period. Of the 38 infected hosts, 32 contained a single worm, 4 hosts contained 2 worms, and 2 hosts contained 3 worms. Five of the hosts with multiple infections contained at least 1 male and 1 female worm. Camel crickets were infected with Neochordodes occidentalis while carabids were infected with an undescribed species of Gordionus . As the majority of hairworms are collected in the post-parasitic adult phase, host data and hairworm-arthropod associations remain poorly documented and our work adds new data to this area of nematomorph biology.     

A rapid detection of circulating antigens of Dirofilaria immitis in dogs by a dot enzyme-linked immunosorbent assay

This report describes a dot enzyme-linked immunosorbent assay (Dot-ELISA) for detecting circulating antigens in the sera of dogs infected with Dirofilaria immitis (D. immitis). Circulating D. immitis antigens could be detected in 24 of 25 infected dogs. The remaining animal had two immature worms. However, non-infected dogs and dogs infected with other parasites were all negative. Few cross-reactions to different parasite antigens were observed. The advantages of the Dot-ELISA include; 1) there is no need for pretreatment and dilution of sera and samples could be immediately bound to nitrocellulose paper set into microfiltration apparatus, 2) this assay could be carried out within 2 h at room temperature, 3) the resulting enzyme-reaction could be measured by both visual observation and densitometric reading.